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Sample records for blood protein electrophoresis

  1. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  2. Red blood cell morphology and plasma proteins electrophoresis of ...

    African Journals Online (AJOL)

    SERVER

    2007-07-04

    Jul 4, 2007 ... The European pond terrapin (Emys orbicularis) is a turtle found in southern and central Europe, West. Asia and North Africa. In this study, we used juvenile E. orbicularis (females of 4 - 5 years old) which was captured from the different area of the Mazandaran province in April 2006. Blood was collected.

  3. Red blood cell morphology and plasma proteins electrophoresis of ...

    African Journals Online (AJOL)

    The European pond terrapin(Emys orbicularis) is a turtle found in southern and central Europe, West Asia and North Africa. In this study, we used juvenile E. orbicularis (females of 4 - 5 years old) which was captured from the different area of the Mazandaran province in April 2006. Blood was collected from the dorsal sinus ...

  4. Protein electrophoresis - urine

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003589.htm Urine protein electrophoresis test To use the sharing features on this page, please enable JavaScript. The urine protein electrophoresis (UPEP) test is used to estimate how much ...

  5. Sex-specific and blood meal-induced proteins of Anopheles gambiae midguts: analysis by two-dimensional gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Laurent-Winter C

    2003-02-01

    Full Text Available Abstract Background Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite. Methods We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood. Results Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females. Conclusion Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity.

  6. Sex-specific and blood meal-induced proteins of Anopheles gambiae midguts: analysis by two-dimensional gel electrophoresis

    Science.gov (United States)

    Prévot, GI; Laurent-Winter, C; Rodhain, F; Bourgouin, C

    2003-01-01

    Background Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite. Methods We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood. Results Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females. Conclusion Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity. PMID:12605724

  7. Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Jessen, Flemming; Wulff, Tune

    2015-01-01

    A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS......-PAGE to be a plasma protein concentration of about 10mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical......-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins...

  8. Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

    International Nuclear Information System (INIS)

    Giometti, C.S.; Willard, K.E.; Anderson, N.L.

    1982-01-01

    Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with 125 I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture

  9. Capillary electrophoresis of proteins for proteomic studies.

    Science.gov (United States)

    Manabe, T

    1999-10-01

    Analyses of proteins in complex mixtures such as cell lyzates are presently performed mainly by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. For structural analysis, each protein in a spot is digested with proteases and the fragment peptides are subjected to Edman sequencing and/or mass spectrometry. These works aim at the total analysis of proteins in a complex mixture and reconstruction of their cooperative functions. Genomic studies are now being combined with these proteomic studies. This review article focuses on the application of capillary electrophoresis aiming at the total analysis of complex protein systems or structural analysis of each separated protein. From this viewpoint, articles on capillary zone electrophoresis, capillary isoelectric focusing, and sieving SDS capillary electrophoresis are reviewed. Since these techniques of capillary electrophoresis have been thoroughly reviewed previously, papers published in 1997 and 1998 are mainly covered.

  10. Fractionation of liver proteins by preparative electrophoresis.

    Science.gov (United States)

    Fountoulakis, M; Juranville, J-F; Tsangaris, G; Suter, L

    2004-02-01

    Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.

  11. Protein blotting with direct blotting electrophoresis.

    Science.gov (United States)

    Beck, S

    1988-05-01

    Direct blotting electrophoresis, a method designed to be of general application for the separation and electroblotting of macromolecules, has been adapted to produce protein blots suitable for subsequent processing by standard techniques such as dye staining or immunological detection. After their separation in a very short gel the protein bands are electrophoresed out of the gel onto an immobilizing matrix. The matrix which is moved across the bottom of the gel by a conveyor belt binds these proteins with high affinity. Once the protein samples have been loaded onto the gel and electrophoresis has been started, no further intervention is needed until the blot is completed. The total expenditure of time for such a direct blot is less than 4 h for a mixture of proteins in the molecular weight range of 14-70 kDa. The staining sensitivity of directly blotted proteins is about 200 ng protein per band as revealed by India ink staining.

  12. CRUDE PROTEIN ELECTROPHORESIS OF SEEDS OF TEN

    African Journals Online (AJOL)

    A A Essiett

    Seeds of mature fruits of ten species of Solanum were collected from the gardens near the screen house, Botany. Department, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. Crude seed proteins were extracted from them and characterised using polyacrylamide gel electrophoresis. Inter and intra specific ...

  13. Microchip analysis of lithium in blood using moving boundary electrophoresis and zone electrophoresis

    NARCIS (Netherlands)

    Vrouwe, E.X.; Lüttge, Regina; Olthuis, Wouter; van den Berg, Albert

    The determination of inorganic cations in blood plasma is demonstrated using a combination of moving boundary electrophoresis (MBE) and zone electrophoresis. The sample loading performed under MBE conditions is studied with the focus on the quantitative analysis of lithium. A concentration

  14. Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.

    Science.gov (United States)

    Browning, Mark; Vanable, Joseph

    2002-01-01

    Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)

  15. Enrichment of low-abundance brain proteins by preparative electrophoresis.

    Science.gov (United States)

    Fountoulakis, Michael; Juranville, Jean François

    2003-02-15

    Detection of low-copy-number gene products is essential for the development of novel drugs, however, it represents a major drawback of proteomics and simultaneously a scientific challenge. We studied the enrichment of rat brain cytosolic proteins by preparative electrophoresis using the PrepCell apparatus. The electrophoresis was performed in the presence of 0.1% lithium dodecyl sulfate. The proteins eluted from the gel were analyzed by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption ionization mass specrometry. Lithium dodecyl sulfate was easily exchanged against agents compatible with isoelectric focusing. Low-abundance proteins, which had not been found before, including neuronal-specific and calcium-binding proteins, were detected. In particular, low-molecular-mass proteins, such as hippocalcin, visinin-like proteins, and 14-3-3 proteins were strongly enriched by preparative electrophoresis.

  16. Hemoglobin electrophoresis

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003639.htm Hemoglobin electrophoresis To use the sharing features on this page, please enable JavaScript. Hemoglobin is a protein that carries oxygen in the blood. Hemoglobin electrophoresis measures the levels of the different types of ...

  17. Study of ABO blood types by combining membrane electrophoresis with surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Wang, Jing; Lin, Juqiang; Huang, Zufang; Sun, Liqing; Shao, Yonghong; Lu, Peng; Shi, Wei; Lin, Jinyong; Chen, Rong

    2012-12-01

    The molecular characterization of ABO blood types, which is clinically significant in blood transfusion, has clinical and anthropological importance. Polymerase chain reaction sequence-based typing (PCR-SBT) is one of the most commonly used methods for the analysis of genetic bases of ABO blood types. However, such methods as PCR-SBT are time-consuming and are high in demand of equipments and manipulative skill. Here we showed that membrane electrophoresis based SERS method employed for studying the molecular bases of ABO blood types can provide rapidand easy-operation with high sensitivity and specificity. The plasma proteins were firstly purified by membrane electrophoresis and then mixed with silver nanoparticles to perform SERS detection. We use this method to classify different blood types, including blood type A (n=13), blood type B (n=9) and blood type O (n=10). Combination of principal component analysis (PCA) and liner discriminant analysis (LDA) was then performed on the SERS spectra of purified albumin, showing good classification results among different blood types. Our experimental outcomes represent a critical step towards the rapid, convenient and accurate identification of ABO blood types.

  18. Development in electrophoresis: instrumentation for two-dimensional gel electrophoresis of protein separation and application of capillary electrophoresis in micro-bioanalysis

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Aoshuang [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    This dissertation begins with a general introduction of topics related to this work. The following chapters contain three scientific manuscripts, each presented in a separate chapter with accompanying tables, figures, and literature citations. The final chapter summarizes the work and provides some prospective on this work. This introduction starts with a brief treatment of the basic principles of electrophoresis separation, followed by a discussion of gel electrophoresis and particularly polyacrylamide gel electrophoresis for protein separation, a summary of common capillary electrophoresis separation modes, and a brief treatment of micro-bioanalysis application of capillary electrophoresis, and ends with an overview of protein conformation and dynamics.

  19. hiv and serum protein electrophoresis patterns in kwazulu

    African Journals Online (AJOL)

    2011-04-01

    Apr 1, 2011 ... To describe the effect of HIV serostatus on serum proteins, serum protein electrophoresis (SPEP) patterns and monoclonal bands. Setting. Inkosi Albert Luthuli Central Hospital, Durban. Design. Retrospective, anonymous analysis of routine laboratory results. Results. Monoclonal bands were not increased ...

  20. Two-dimensional polyacrylamide gel electrophoresis of intracellular proteins

    International Nuclear Information System (INIS)

    Ojima, N.; Sakamoto, T.; Yamashita, M.

    1996-01-01

    Since two-dimensional electrophoresis was established by O'Farrell for analysis of intracellular proteins of Escherichia coli, it has been applied to separation of proteins of animal cells and tissues, and especially to identification of stress proteins. Using this technique, proteins are separated by isoelectric focusing containing 8 m urea in the first dimension and by SDS-PAGE in the second dimension. The gels are stained with Coomassie Blue R-250 dye, followed by silver staining. In the case of radio-labeled proteins, the gels are dried and then autoradiographed. In order to identify a specific protein separated by two-dimensional electrophoresis, a technique determining the N-terminal amino acid sequence of the protein has been developed recently. After the proteins in the gel were electrotransferred to a polyvinylidene difluoride membrane, the membrane was stained for protein with Commassie Blue and a stained membrane fragment was applied to a protein sequencer. Our recent studies demonstrated that fish cells newly synthesized various proteins in response to heat shock, cold nd osmotic stresses. For example, when cellular proteins extracted from cold-treated rainbow trout cells were subjected to two-dimensional gel electrophoresis, the 70 kDa protein was found to be synthesized during the cold-treatment. N-Terminal sequence analysis showed that the cold-inducible protein was a homolog of mammalian valosin-containing protein and yeast cell division cycle gene product CDC48p. Furthermore, the sequence data were useful for preparing PCR primers and a rabbit antibody against a synthetic peptide to analyze a role for the protein in the function of trout cells and mechanisms for regulation

  1. Serum Protein Electrophoresis: Any role in monitoring for ...

    African Journals Online (AJOL)

    Background: Developing world are always looking for monitoring tools during reagent shortage and equipments troubles which are very frequent. The aim of this study was to evaluate Serum Protein Electrophoresis (SPE) as a marker for assessing HIV treatment response. Methods: A cross-sectional study was conducted ...

  2. Capillary zone electrophoresis-mass spectromet of intact proteins

    NARCIS (Netherlands)

    Domínguez-Vega, Elena; Haselberg, Rob; Somsen, Govert W.

    2016-01-01

    Capillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS

  3. Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis.

    Science.gov (United States)

    Duncombe, Todd A; Herr, Amy E

    2013-06-07

    Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. For the protein electrophoresis study described here, fsPAGE lanes are comprised of a sample reservoir and contiguous separation gel. No enclosed microfluidic channels are employed. The fsPAG devices (120 μm tall) are directly photopatterned atop of and covalently attached to planar polymer or glass surfaces. Leveraging the fast uses readily available materials and instruments - making this technique highly accessible.

  4. Attempt to run urinary protein electrophoresis using capillary technique.

    Science.gov (United States)

    Falcone, Michele

    2014-10-01

    The study of urinary protein has a predominant place in the diagnosis of kidney disease. The most common technique is agarose gel electrophoresis (AGE). For several years, the technique of choice applied to the analysis of serum proteins has been CE, a system that uses capillary fused silica, subjected to high voltage to separate and measure serum proteins. The purpose of this paper was to perform capillary electrophoresis on urinary proteins which, at present, are not interpretable due to the many nonspecific peaks visible when using gel electrophoresis. In order to carry out our research, we used a capillary V8 analyzer together with an agarose gel system from the same company. AGE was taken as the reference method, for which urine was used without any pretreatment. For the V8 system, urine was subjected to purification on granular-activated carbon and then inserted into the V8 analyzer, selecting a program suitable for liquids with low protein content. We examined 19 urine samples collected over 24 hrs from both hospitalized and external patients with different types of proteinuria plus a serum diluted 1/61 considered as a control to recognize the bands. Both methods showed the same protein fractions and classified the proteinuria in a similar way. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Plasma protein electrophoresis of Trachemys scripta and Iguana iguana.

    Science.gov (United States)

    Giménez, Mercè; Saco, Yolanda; Pato, Raquel; Busquets, Alex; Martorell, Jaime M; Bassols, Anna

    2010-06-01

    Protein electrophoresis is widely applied in veterinary medicine, but is not used often in reptiles, in part because of lack of reference values. The goals of this study were to compare plasma protein profiles obtained by cellulose acetate electrophoresis (CAE) and agarose gel electrophoresis (AGE), measure precision and examine interference by sample hemolysis, and establish preliminary reference intervals for 2 reptile species. Heparinized plasma samples from healthy and diseased adult female Iguana iguana (n=40) and Trachemys scripta (n=60) were analyzed by CAE and AGE. Total protein concentration was measured by the biuret method. Electrophoresis results were compared using Bland-Altman plots and Passing-Bablok regression analysis. Precision and the effects of sample hemolysis were determined. Results from clinically healthy animals were used to determine reference intervals. Five protein fractions were identified in both species, with bisalbuminemia observed in 23/40 iguanas. High correlation was observed between the 2 methods for all fractions, with few proportional and systematic errors. Coefficients of variation were lower using AGE vs CAE and for I. iguana vs T. scripta. Two additional bands were observed in hemolyzed samples from T. scripta; 1 additional band was observed for I. iguana. Minimum and maximum values were reported for healthy I. iguana (n=14) and T. scripta (n=22). Although both methods are acceptable, the performance of AGE was slightly better than that of CAE for analysis of plasma from reptiles. Furthermore, reptile electrophoretic patterns should be interpreted based on the method used, the species analyzed, and the quality of the plasma sample.

  6. Serum Protein Electrophoresis in Dogs With Intestinal Parasites

    OpenAIRE

    KAYMAZ, Alev AKDOĞAN; BAKIREL, Utku; GÖNÜL, Remzi; TAN, Hüseyin

    1999-01-01

    The serum of 66 dogs with intestinal parasites (showing gastrointestinal problems caused by taeniosis, coccidiosis, ancylostomosis, trichuriosis and ascarididosis) was examined by electrophoresis. There were 6 dogs with coccidiosis, 6 dogs with ancylostomosis, 6 dogs with trichuriosis, 24 dogs with taeniosis and 24 dogs with ascarididosis. After agar gel protein electorphoresis of the serum samples, ?1 globulin levels were significantly lower in the coccidiosis group than in the other grou...

  7. Procedures for two-dimensional electrophoresis of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tollaksen, S.L.; Giometti, C.S.

    1996-10-01

    High-resolution two-dimensional gel electrophoresis (2DE) of proteins, using isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the second, was first described in 1975. In the 20 years since those publications, numerous modifications of the original method have evolved. The ISO-DALT system of 2DE is a high-throughput approach that has stood the test of time. The problem of casting many isoelectric focusing gels and SDS-PAGE slab gels (up to 20) in a reproducible manner has been solved by the use of the techniques and equipment described in this manual. The ISO-DALT system of two-dimensional gel electrophoresis originated in the late 1970s and has been modified many times to improve its high-resolution, high-throughput capabilities. This report provides the detailed procedures used with the current ISO-DALT system to prepare, run, stain, and photograph two-dimensional gels for protein analysis.

  8. Direct measurement of lithium in whole blood using microchip capillary electrophoresis with integrated conductivity detection

    NARCIS (Netherlands)

    Vrouwe, E.X.; Lüttge, Regina; van den Berg, Albert

    2004-01-01

    The direct measurement of lithium in whole blood is described. Using microchip capillary electrophoresis (CE) with defined sample loading and applying the principles of column coupling, alkali metals were determined in a drop of whole blood. Blood collected from a finger stick was mixed with

  9. The Cutoff Level for Urine Protein in Urine Immunofixation Electrophoresis.

    Science.gov (United States)

    Ellidag, Hamit Yasar; Curek, Gulten; Eren, Esin; Aydin, Ozgur; Yilmaz, Necat

    2015-01-01

    Immunofixation electrophoresis (IFE) maintains its importance in diagnosing monoclonal gammopathies. In particular, urine IFE detects free light chains (FLC) in urine samples even at low concentrations and offers higher sensitivity compared to serum electrophoresis and serum IFE. The aim of the present study was to determine the place and significance of quantitative urinary protein measurement before IFE in interpreting the results of subsequent IFE and to determine the most appropriate protein concentrations for the appearance of bands. The records of a total of 600 patients, who underwent screening for Bence Jones proteinuria using IFE on 24-hour urine, were retrospectively reviewed. Urine IFE was performed using Helena SAS-I and SAS-I devices. The total protein concentration in the urine was quantitatively determined by the Pyrogallol red method, and the urine albumin level was determined using the immunoturbidimetric method. These analyses were measured on an Olympus/Beckmann AU5800. The evaluation of IFE results revealed that 311 patients had normal results, 108 patients had monoclonal bands, five patients had biclonal bands, 28 had polyclonal bands, and 148 patients had various degrees of proteinuria. ROC curves were created in order to determine the most appropriate urinary protein and albumin levels to observe bands in IFE. Accordingly, urine baseline protein level (mg/dL) showed the highest AUC value (cutoff value: 19.4 mg/dL, sensitivity: 92%, specificity: 98.2%, AUC: 0.972). The present study showed that quantitative protein measurement before IFE eliminated the disadvantages associated with the IFE method and its interpretation.

  10. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

    Science.gov (United States)

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J

    2014-09-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

  11. Protein Mobility Shifts Contribute to Gel Electrophoresis Liquid Chromatography Analysis.

    Science.gov (United States)

    Carruthers, Nicholas J; Parker, Graham C; Gratsch, Theresa; Caruso, Joseph A; Stemmer, Paul M

    2015-09-01

    Profiling of cellular and subcellular proteomes by liquid chromatography with tandem mass spectrometry (MS) after fractionation by SDS-PAGE is referred to as GeLC (gel electrophoresis liquid chromatography)-MS. The GeLC approach decreases complexity within individual MS analyses by size fractionation with SDS-PAGE. SDS-PAGE is considered an excellent fractionation technique for intact proteins because of good resolution for proteins of all sizes, isoelectric points, and hydrophobicities. Additional information derived from the mobility of the intact proteins is available after an SDS-PAGE fractionation, but that information is usually not incorporated into the proteomic analysis. Any chemical or proteolytic modification of a protein that changes the mobility of that protein in the gel can be detected. The ability of SDS-PAGE to resolve proteins with chemical modifications has not been widely utilized within profiling experiments. In this work, we examined the ability of the GeLC-MS approach to help identify proteins that were modified after a small hairpin RNA-dependent knockdown in an experiment using stable isotope labeling by amino acids in cell culture-based quantitation.

  12. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population

    DEFF Research Database (Denmark)

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine

    2014-01-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum...... proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type....... Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations...

  13. THE USE OF ELECTROPHORESIS IN LABORATORY DIAGNOSTIC OF SERUM PROTEINS IN VETERINARY MEDICINE

    Directory of Open Access Journals (Sweden)

    Veronika Nagyová

    2013-06-01

    Full Text Available In this study, we explain the need for more detailed laboratory diagnostic of serum proteins in veterinary medicine. The application of proteomic approaches over the last decade has provided new tools for clarifying the molecular aspects of physiological states, and for understanding the etiology and pathogenesis of many diseases. Proteomics performs large-scale protein analysis, describes the entire protein complement of a cell, tissue, biological fluid or organism and provides information on protein expression, localization, functions and interactions. Majority of plasmatic proteins is synthesized in hepatocytes, with albumin representing their largest quantitative part. The second major contributor is the imune system. Plasma proteins perform a nutritive function, exert colloidal osmotic pressure, and aid in the maintenance of acid-base balance. Individual proteins serve as enzymes, antibodies, coagulation factors, hormones, and transport substances. Fresh serum contains all of the plasma proteins except fibrinogen, factor V, and factor VIII. These are consumed during clot formation. The electrophoretic technique is the current standard of reference for the fractionation of the serum proteins in clinical biochemistry. Serum proteins determined by electrophoresis involves albumin, α1, α2, β1, β2 and γ-globulins. Individual blood serum proteins have different functions and their identification is used also as a diagnostic tool. Many of these proteins are the so-called acute phase proteins. For example, α1-acid glycoprotein concentration increases during inflammation or infection, as well as concentrations of other acute phase proteins (CRP, serum amyloid A protein, α1-antitrypsin, haptoglobin, ceruloplasmin and fibrinogen based on increased synthesis of hepatocytes with subsequent release of these proteins in blood. Alfa1-antitrypsin, member of the superfamily of proteinase inhibitors, has a crucial effect on inactivation of neutrophil

  14. Protein expression on Cr resistant microorganism using electrophoresis method

    Directory of Open Access Journals (Sweden)

    SAJIDAN

    2009-01-01

    Full Text Available Fatmawati U, Suranto, Sajidan. 2009. Protein expression on Cr resistant microorganism using electrophoresis method. Nusantara Bioscience 1: 31-37. Hexavalent chromium (Cr(VI is known as toxic heavy metals, so the need is reduced to Cr(III is much less toxicity. Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumoniae, Pantoea sp. and Saccharomyces cerevisiae are resistant Cr(VI microorganism and have ability to reduce Cr(VI. The aim of this research is to know ability of microorganism to reduce Cr(VI and to know protein band pattern between Cr(VI resistant microorganism and non resistant microorganism which inoculated on LB broth. SDS-PAGE was used to indentify protein expression. While, Cr(VI concentration was identified by 1.5 diphenylcarbazide method. The quantitative data was analyzed by two factorial ANOVA that continued with DMRT at 1% level test. The qualitative data i.e. protein expression analyzed by relative mobility (Rf. The results showed that the ability of microorganisms to reduce Cr(VI at initial concentration of 0.5 ppm, 1 ppm, 5 ppm and 10 ppm may vary, the average percentage of the ability of each microorganism in reducing Cr(VI is P. putida (65% > S. cerevisiae (64.45% >. P. aeruginosa (60.73% > Pantoea sp. (50.22% > K. pneumoniae (47.82% > without microorganisms (34.25%. The adding microorganisms have significantly influenced toward reduction of Cr(VI. The SDS-PAGE shows that protein expression between resistant and not resistant microorganisms are no different, but resistant microorganisms have more protein (protein band is thicker.

  15. Immunoelectrophoresis - blood

    Science.gov (United States)

    IEP - serum; Immunoglobulin electrophoresis - blood; Gamma globulin electrophoresis; Serum immunoglobulin electrophoresis; Amyloidosis - electrophoresis serum; Multiple myeloma - serum electrophoresis; Waldenström - serum electrophoresis

  16. Recognition and management of common, rare, and novel serum protein electrophoresis and immunofixation interferences

    DEFF Research Database (Denmark)

    McCudden, Christopher R; Jacobs, Joannes F M; Keren, David

    2018-01-01

    Protein electrophoresis and immunofixation are subject to a variety of analytical interferences that may affect monoclonal protein diagnostics performed in the context of monoclonal gammopathies. Interferences include endogenous substances, such as hemoglobin and fibrinogen, and exogenous compounds...

  17. Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting

    DEFF Research Database (Denmark)

    Andersen, H; Birkelund, Svend; Christiansen, Gunna

    1987-01-01

    The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others...... isolated variously from genital tract, mouth, blood, upper urinary tract and a wound. These 14 strains shared 76-99% of proteins in SDS-gradient gel analysis and 41-72% in the 2D gels. As expected, the immunoblot analysis likewise revealed the existence of an extensive common protein pattern in M. hominis...

  18. Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique.

    Science.gov (United States)

    Gama, Fernanda Gomes Velasque; Santana, Aureo Evangelista; Filho, Eugênio de Campos; Nogueira, Cláudia Aparecida da Silva

    2007-03-01

    Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.

  19. Protein C blood test

    Science.gov (United States)

    ... in the body that prevents blood clotting. A blood test can be done to see how much of ... Certain medicines can change blood test results. Tell your health care provider about all the medicines you take. Your provider will tell you if you need ...

  20. Protein S blood test

    Science.gov (United States)

    ... in your body that prevents blood clotting. A blood test can be done to see how much of ... Certain medicines can change blood test results: Tell your health care provider about all the medicines you take. Your provider will tell you if you need ...

  1. Two-dimensional electrophoresis protein profiling as an analytical tool for human acute leukemia classification.

    Science.gov (United States)

    Cui, Jiu-Wei; Wang, Jie; He, Kun; Jin, Bao-Feng; Wang, Hong-Xia; Li, Wei; Kang, Li-Hua; Hu, Mei-Ru; Li, Hui-Yan; Yu, Ming; Shen, Bei-Fen; Wang, Guan-Jun; Zhang, Xue-Min

    2005-01-01

    Two-dimensional electrophoresis (2-DE) was used to profile the proteins of leukemic cells from 61 cases of akute leukemia (AL) characterized by the French-American-British (FAB) classification. The differentially expressed protein spots were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). The distinct protein profiles (DPPs) of AL FAB subtypes were explored successfully, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5), and acute lymphoid leukemia (ALL), which were homogeneous within different samples of the same subgroup but clearly differed from all other subgroups. We also found a group of proteins differentially expressed between AL cells and normal white blood cells. Among the DPPs of AL subtypes, some proteins have been reported, but most of them were first reported here to mark AML differentiation and to discriminate AML from ALL. These data show that 2-DE protein profiling could be used as an analytical tool for facilitating molecular definition of human AL classification and understanding the mechanism of leukemogensis, and the extension of the present analysis to the currently less well-defined AL will identify additional subgroups and may promote the identification of new targets for specific treatment approaches.

  2. Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes.

    Science.gov (United States)

    Dowdle, Megan E; Imboden, Susanne Blaser; Park, Sookhee; Ryder, Sean P; Sheets, Michael D

    2017-07-28

    Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

  3. A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins.

    OpenAIRE

    Tastet, Christophe; Lescuyer, Pierre; Diemer, Hélène; Luche, Sylvie; Van Dorsselaer, Alain; Rabilloud, Thierry

    2003-01-01

    website publisher www.interscience.wiley.com; International audience; A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300 000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above M(r) range. The buffer system uses taurine and chloride as trailing and leading ion, re...

  4. Type 2 diabetes mellitus with early phase acute inflammatory protein on serum protein electrophoresis

    Directory of Open Access Journals (Sweden)

    ET Tuladhar

    2012-03-01

    Full Text Available Background: The onset of Type 2 diabetes has been associated with low grade systemic inflammation. The inflammatory status has been studied by measuring acute phase reactant proteins like hsCRP, α1- antitrypsin, α1-acid glycoprotein, ceruloplasmin, fibrinogen. Most of these acute phase reactants form α1 and α2 bands on electropherogram of serum proteins. The aim of this study was to evaluate inflammatory status in controlled and uncontrolled type 2 diabetes using cellulose acetate electrophoresis and to find the impact of glycemic status as indicated by HbA1c on inflammation process. Materials and Methods: Serum protein electrophoresis was done on serum samples of 60 cases of Diabetes [controlled and uncontrolled] using cellulose acetate paper technique. The electropherogram obtained was stained with Ponseu S and then quantitated using densitometer. Glycemic status was studied by HbA1c analysis. The density of α1and α2 bands in electropherogram were correlated with HbA1c level. Result: A significant increase in the percentage of α1 and α2 band proteins (0.765 and 0.716, p<0.001 were found with the increasing level of HbA1c. With cutoff of HbA1c 7% (American Diabetic Association recommended, the α1 and α2 serum proteins concentration are significantly higher (p<0.001 in uncontrolled diabetes mellitus compared to controlled diabetes mellitus Conclusion: Cellulose acetate electrophoresis of serum proteins show early phase acute inflammatory status in uncontrolled type 2 diabetes mellitus. The process of systemic inflammation worsens with uncontrolled glycemia as indicated by HbA1c. Inflammatory status should be studied adjunct to glycemic status. DOI: http://dx.doi.org/10.3126/jpn.v2i3.6024 JPN 2012; 2(3: 211-214

  5. Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis.

    Science.gov (United States)

    Danish, Azeem; Lee, Sang-Yong; Müller, Christa E

    2017-10-07

    A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A 2A receptor (A 2A AR), and connexin 43 (Cx43). Quantification of SV2A and A 2A AR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

  6. Cetyltrimethylammonium bromide discontinuous gel electrophoresis: Mr-based separation of proteins with retention of enzymatic activity.

    Science.gov (United States)

    Akins, R E; Levin, P M; Tuan, R S

    1992-04-01

    A discontinuous polyacrylamide and agarose gel electrophoresis system is presented here which allows the fine separation of proteins based on molecular weight with the concomitant retention of native enzymatic activity. This system, referred to as the CAT gel, uses the cationic detergent cetyltrimethylammonium bromide (CTAB) and includes a stacking gel based on the zwitterion arginine and the buffer N-tris(hydroxymethyl)-methylglycine. The CAT gel system allows specific enzyme histochemical detection and localization of proteins after gel electrophoresis. We present evidence that the CAT system stacked and separated a broad range of proteins into discrete bands which migrate as a linear function of log Mr. We have also assessed the effect of CTAB solubilization on the activity of several proteins and showed that some proteins separated by CAT electrophoresis maintain high levels of native enzymatic activity and may be detected histochemically in situ.

  7. Preparation of Barley Storage Protein, Hordein, for Analytical Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    DEFF Research Database (Denmark)

    Doll, Hans; Andersen, Bente

    1981-01-01

    The extraction, reduction, and alkylation of barley hordein for routine electrophoresis in sodium dodecyl sulfate-polyacrylamide gels were studied to set up a simple preparation procedure giving well-resolved bands in the electrophoresis gel. Hordein was extracted from single crushed seeds or flour...... by aqueous 50% propan-2-ol containing a Tris-borate buffer, pH 8.6. The presence of the buffer facilitates the consecutive complete reduction of the extracted protein in the alcohol. Reduction and alkylation in the buffer containing propan-2-ol give sharper bands in the electrophoresis than reduction...

  8. A simple cellulose acetate membrane-based small lanes technique for protein electrophoresis.

    Science.gov (United States)

    Na, Na; Liu, Tingting; Yang, Xiaojun; Sun, Binjie; Ouyang, Jenny; Ouyang, Jin

    2012-08-01

    Combining electrophoresis with a cellulose acetate membrane-based technique, we developed a simple and low-cost method, named cellulose acetate membrane-based small lanes (CASL), for protein electrophoresis. A home-made capillary plotter controlled by a 3D moving stage was used to create milli-to-micro channels by printing poly(dimethylsiloxane) on to a hydrophilic cellulose acetate membrane. In the hydrophilic channels, 5 nL protein mixture was separated on the basis of electro-migration under an electric field. Compared with polyacrylamide gel electrophoresis (PAGE), CASL resulted in higher protein signal intensity for separation of mixtures containing the same mass of protein. The platform was easily fabricated at low cost (approx. $0.005 for each 1-mm-wide channel), and separation of three protein mixtures was completed in 15 min. Both electrophoresis time and potential affected the separation. Rather than chromatographic separation, this method accomplished application of microchannel techniques for cellulose acetate membrane-based protein electrophoresis. It has potential in proteomic analysis, especially for rapid, low-cost, and low-volume sample analysis in clinical diagnosis.

  9. Analysis of Soluble Proteins in NaturalCordyceps sinensisfrom Different Producing Areas by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Two-dimensional Electrophoresis.

    Science.gov (United States)

    Li, Chun-Hong; Zuo, Hua-Li; Zhang, Qian; Wang, Feng-Qin; Hu, Yuan-Jia; Qian, Zheng-Ming; Li, Wen-Jia; Xia, Zhi-Ning; Yang, Feng-Qing

    2017-01-01

    As one of the bioactive components in Cordyceps sinensis (CS), proteins were rarely used as index components to study the correlation between the protein components and producing areas of natural CS. Protein components of 26 natural CS samples produced in Qinghai, Tibet, and Sichuan provinces were analyzed and compared to investigate the relationship among 26 different producing areas. Proteins from 26 different producing areas were extracted by Tris-HCl buffer with Triton X-100, and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). The SDS-PAGE results indicated that the number of protein bands and optical density curves of proteins in 26 CS samples was a bit different. However, the 2-DE results showed that the numbers and abundance of protein spots in protein profiles of 26 samples were obviously different and showed certain association with producing areas. Based on the expression values of matched protein spots, 26 batches of CS samples can be divided into two main categories (Tibet and Qinghai) by hierarchical cluster analysis. The number of protein bands and optical density curves of proteins in 26 Cordyceps sinensis samples were a bit different on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profilesNumbers and abundance of protein spots in protein profiles of 26 samples were obvious different on two-dimensional electrophoresis mapsTwenty-six different producing areas of natural Cordyceps sinensis samples were divided into two main categories (Tibet and Qinghai) by Hierarchical cluster analysis based on the values of matched protein spots. Abbreviations Used : SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2-DE: Two-dimensional electrophoresis, Cordyceps sinensis : CS, TCMs: Traditional Chinese medicines.

  10. Isolation of monodisperse nanodisc-reconstituted membrane proteins using free flow electrophoresis

    DEFF Research Database (Denmark)

    Justesen, Bo Højen; Laursen, Tomas; Weber, Gerhard

    2013-01-01

    Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e...

  11. Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

    NARCIS (Netherlands)

    Heuberger, E.H M L; Veenhoff, L.M.; Duurkens, R.H.T.; Friesen, R.H.E.; Poolman, B.

    2002-01-01

    Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By

  12. HIV and serum protein electrophoresis patterns in KwaZulu-Natal: a ...

    African Journals Online (AJOL)

    Objective. To describe the effect of HIV serostatus on serum proteins, serum protein electrophoresis (SPEP) patterns and monoclonal bands. Setting. Inkosi Albert Luthuli Central Hospital, Durban. Design. Retrospective, anonymous analysis of routine laboratory results. Results. Monoclonal bands were not increased in ...

  13. Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry

    OpenAIRE

    Singh, Pramod Kumar; Shrivastava, Nidhi; Chaturvedi, Krishna; Sharma, Bechan; Bhagyawant, Sameer S.

    2016-01-01

    Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE) across a broad range 3.0–10.0 immobilized pH gradient (IPG) strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected ...

  14. Capillary electrophoresis coupled to fluorescence spectroscopy for protein characterisation

    NARCIS (Netherlands)

    de Kort, B.J.

    2012-01-01

    Proteins are essential molecules in all living organisms. Their involvement in numerous biological processes has led to the development of protein-based medicines (biopharmaceuticals). For good understanding of the properties and function of endogenous proteins and biopharmaceuticals, extensive

  15. Variations of plasma protein electrophoresis in healthy captive Green Iguanas (Iguana iguana).

    Science.gov (United States)

    Musilová, Anna; Knotková, Zora; Pinterová, Kateřina; Knotek, Zdeněk

    2015-06-01

    Serum or plasma protein electrophoresis is used as a routine test for health assessment in veterinary medicine, but there are only a limited number of studies regarding clinical use of electrophoresis in reptile species. The goals of this study were to establish reference intervals for plasma protein electrophoresis in the Green Iguana (Iguana iguana), compare values between males and females, and to identify season-related changes. Plasma samples were obtained from 21 healthy captive male and female Green Iguanas. Agarose gel electrophoresis was performed using an automated Hydrasys system. Four main protein fractions were observed: albumin, α globulins, β globulins, and γ globulins. Bisalbuminemia was observed in 4 of 21 healthy iguanas. Minimum and maximum values were reported for healthy Green Iguanas in March, June, September, and December. Seasonal changes in albumin were determined between March and December, and in γ globulins between June and September. Differences between males and females were seen in albumin concentration in September. Reference intervals of the plasma protein fractions according to electrophoresis in the Green Iguana can be affected by seasonal changes and sex of animals. It should be taken into account when clinical evaluation is performed. © 2015 American Society for Veterinary Clinical Pathology.

  16. Modification of resolution in capillary electrophoresis for protein profiling in identification of genetic modification in foods

    OpenAIRE

    Latoszek, A.; Cifuentes, Alejandro

    2011-01-01

    The capillary electrophoresis with UV detection was employed for protein profiling in extracts from maize and soybeans. Modifications of back-ground electrolyte and coating the capillary wall with polybrene was employed in order to decrease the protein adsorption on the capillary walls. The obtained protein profiles were compared for transgenic and non-transgenic variants, showing in some cases significant changes that might be employed for identification of genetic modifications ...

  17. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

    Science.gov (United States)

    Belov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.

    2017-12-01

    Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). [Figure not available: see fulltext.

  18. Monoclonal protein reference change value as determined by gel-based serum protein electrophoresis.

    Science.gov (United States)

    Salamatmanesh, Mina; McCudden, Christopher R; McCurdy, Arleigh; Booth, Ronald A

    2018-01-01

    The International Myeloma Working Group recommendations for monitoring disease progression or response include quantitation of the involved monoclonal immunoglobulin. They have defined the minimum change criteria of ≧25% with an absolute change of no gel-based serum protein electrophoresis. Sixteen clinically stable MGUS patients were identified from our clinical hematology database. Individual biological variability (CVi) was determined and used to calculate a monoclonal protein reference change value (RCV). Analytical variability of the normal protein fractions (albumin, alpha-1, alpha-2, beta, total gamma) ranged from 1.3% for albumin to 5.8% for the alpha-1 globulins. CVa of low (5.6g/L) and high (32.2g/L) concentration monoclonal proteins were 3.1% and 22.2%, respectively. Individual CVi of stable patients ranged from 3.5% to 24.5% with a CVi of 12.9%. The reference change value (RCV) at a 95% probability was determined to be 36.7% (low) 39.6% (high) using our CVa and CVi. Serial monitoring of monoclonal protein concentration is important for MGUS and multiple myeloma patients. Accurate criteria for interpreting a change in monoclonal protein concentration are required for appropriate decision making. We used QC results and real-world conditions to assess imprecision of serum protein fractions including low and high monoclonal protein fractions and clinically stable MGUS patients to determine CVi and RCV. The calculated RCVs of 36.7% (low) and 39.6% (high) in this study were greater that reported previously and greater than the established criteria for relapse. Response criteria may be reassessed to increase sensitivity and specificity for detection of response. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  19. [Total protein analysis by two-dimensional electrophoresis in cysticerci of Taenia solium and Taenia asiatica].

    Science.gov (United States)

    Fang, Wen; Xiao, Liang-Liang; Bao, Huai-En; Mu, Rong

    2011-06-01

    Two 20-day-old three-way crossed hybrid pigs were infected with 80000 Taenia solium or T. asiatica eggs, respectively. Immature cysticerci of the two species in liver were collected at 40 days after infection. The total proteins were separated by two-dimensional electrophoresis, and differentially expressed proteins were analyzed by Image-Master 2D Platinum 6.0 software. The results showed that there were (236 +/- 12) and (231 +/- 14) protein spots in 2D electrophoresis gel images of T. solium and T. asiatica, respectively, with 3 proteins up-regulated and 7 proteins down-regulated in T. solium cysticercus by 2-fold or more compared with those in T. asiatica cysticercus.

  20. Reference interval determination of hemoglobin fractions in umbilical cord and placental blood by capillary electrophoresis.

    Science.gov (United States)

    Bó, Suzane Dal; de Oliveira Lemos, Fabiane Kreutz; Pedrazzani, Fabiane Spagnol; Cagliari, Cláudia Rosa; Scotti, Luciana

    2016-04-01

    Umbilical cord and placental blood (UCPB) is a rich source of hematopoietic stem cells widely used to treat diseases that did not have effective treatments until recently. Umbilical cord and placental blood banks (UCPBBs) are needed to be created to store UCPB. UCPB is collected immediately after birth, processed, and frozen until infusion. Detection of abnormal hemoglobins is one of UCPB screening tests available. The objective of the present study was to determine the reference interval for HbA, HbF, and HbA2 in UCPB using capillary electrophoresis. Methods: Observational retrospective study of UCPB samples undergoing hemoglobin electrophoresis was performed between April 2012 and May 2013. We analyzed 273 UCPB samples. All cords met the criteria of BrasilCORD. We found 19.9% (10.5–36.7%) for HbA, 80.1% (62.7–89.4%) for HbF, and 0.1% (0.0–0.6%) for HbA2. Data were expressed as median (P2.5–P97.5). Establishing specific reference intervals is the best option for most tests because such ranges reflect the status of the population in which the tests will be applied. The use of appropriate reference intervals ensures that clinical labs provide reliable information, thus enabling clinicians to correctly interpret results and choose the best approach for the target population.

  1. Two Dimensional Electrophoresis of Proteins from Cultures of Erysiphe graminis f.sp. hordei

    DEFF Research Database (Denmark)

    Torp, J.; Andersen, Brian

    1982-01-01

    Conidial proteins from barley powdery mildew, Erysiphe graminis f. sp. hordei, were separated by 2-dimensional electrophoresis in polyacrylamide slab gels. Isoelectric focusing was used in the first dimension and separation according to molecular weight in a gel containing sodium dodecyl sulphate...

  2. Transient isotachophoresis in carrier ampholyte-based capillary electrophoresis for protein analysis

    Czech Academy of Sciences Publication Activity Database

    Busnel, J. M.; Descroix, S.; Godfrin, D.; Hennion, M. C.; Kašička, Václav; Peltre, G.

    2006-01-01

    Roč. 27, č. 18 (2006), s. 3591-3598 ISSN 0173-0835 Institutional research plan: CEZ:AV0Z40550506 Keywords : carrier ampholyte-based capillary electrophoresis * transient isotachophoresis * proteins Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.101, year: 2006

  3. Evaluation of phytic acid as a buffer additive for the separation of proteins in capillary electrophoresis.

    NARCIS (Netherlands)

    Veraart, J.R.; Schouten, Y.; Gooijer, C.; Lingeman, H.

    1997-01-01

    The use of phytic acid to improve protein analysis by capillary electrophoresis (CE) is becoming more and more popular. Due to its size and number of negative charges (up to 12) it provides a high ionic strength combined with a low conductance resulting in an efficient decrease of wall adsorption

  4. Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins

    Science.gov (United States)

    Kim, Thomas D.; Craig, Paul A.

    2010-01-01

    Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

  5. Crude protein electrophoresis of seeds of ten species of Solanum L ...

    African Journals Online (AJOL)

    Seeds of mature fruits of ten species of Solanum were collected from the gardens near the screen house, Botany Department, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. Crude seed proteins were extracted from them and characterised using polyacrylamide gel electrophoresis. Inter and intra specific ...

  6. Native Red Electrophoresis - A new method suitable for separation of native proteins

    Czech Academy of Sciences Publication Activity Database

    Dráb, T.; Kračmerová, J.; Tichá, I.; Hanzlíková, E.; Tichá, M.; Ryšlavá, H.; Doubnerová, V.; Maňásková-Postlerová, Pavla; Liberda, J.

    2011-01-01

    Roč. 32, č. 24 (2011), s. 3597-3599 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA303/09/1285 Institutional research plan: CEZ:AV0Z50520701 Keywords : native electrophoresis * Ponceau RR * Ponceau S * protein Subject RIV: CE - Biochemistry Impact factor: 3.303, year: 2011

  7. Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins

    NARCIS (Netherlands)

    Eriksson, Jonas H.C.; Mol, Roelof; Somsen, Govert W.; Hinrichs, Wouter L.J.; Frijlink, Henderik W.; de Jong, Gerhardus J.

    2004-01-01

    The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium

  8. Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins.

    NARCIS (Netherlands)

    Hoffert, J.D.; Balkom, B.W.M. van; Chou, C.L.; Knepper, M.A.

    2004-01-01

    In this study, we present a standardized approach to purification of native inner medullary collecting duct (IMCD) cells from rat kidney for proteomic analysis and apply the approach to identification of abundant proteins utilizing two-dimensional difference gel electrophoresis (DIGE) coupled with

  9. Comparison of multi-locus enzyme and protein gel electrophoresis ...

    African Journals Online (AJOL)

    Results indicate that the estimated inter-specific variation may be more pronounced with protein markers than with isoyzmes when the two approaches are applied to the same populations. The level of genetic variability detected within and between Fusarium spp. accessions with protein and esterase isoyzmes analysis ...

  10. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Madsen, P S; Hokland, M; Ellegaard, J

    1988-01-01

    We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific MoAbs, and ......We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific Mo...... markers were observed in all cell types. All the putative protein markers have been identified in the protein database of human peripheral blood mononuclear cells (PBMCs) (see accompanying article by Celis et al.). Comparison of the overall patterns of protein synthesis of the unsorted PBMCs with those...

  11. Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

    Science.gov (United States)

    Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

    2011-03-01

    We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

  12. Increase in local protein concentration by field-inversion gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Paulus Aran

    2007-09-01

    Full Text Available Abstract Background Proteins that migrate through cross-linked polyacrylamide gels (PAGs under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE. Results Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS, which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. Conclusion The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein

  13. Increase in local protein concentration by field-inversion gel electrophoresis.

    Science.gov (United States)

    Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-Pui Paul; Leung, Hon-Chiu Eastwood

    2007-09-26

    Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

  14. Determination of fluoroacetate and fluoride in blood serum by capillary zone electrophoresis using capacitively coupled contactless conductivity detection.

    Science.gov (United States)

    Vidal, Denis Tadeu Rajh; Augelli, Marcio Antonio; Hotta, Guilherme Minoru; Lopes, Fernando Silva; do Lago, Claudimir Lucio

    2011-04-01

    Fluoroacetate is a highly toxic species naturally found in plants and in commercial products (compound 1080) for population control of several undesirable animal species. However, it is non-selective and toxic to many other animals including humans, and thus its detection is very important for forensic purposes. This paper presents a sensitive and fast method for the determination of fluoroacetate in blood serum using capillary electrophoresis with capacitively coupled contactless conductivity detection. Serum blood samples were treated with ethanol to remove proteins. The samples were analyzed in BGE containing 15 mmol/L histidine and 30 mmol/L gluconic acid (pH 3.85). The calibration curve was linear up to 75 μmol/L (R² =0.9995 for N=12). The detection limit in the blood serum was 0.15 mg/kg, which is smaller than the lethal dose for humans and other animals. Fluoride, a metabolite of the fluoroacetate defluorination, could also be detected for levels greater than 20 μmol/L, when polybrene was used for reversion of the EOF. CTAB and didecyldimethylammonium bromide are not useful for this task because of the severe reduction of the fluoride level. However, no interference was observed for fluoroacetate. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Bargain Electrophoresis.

    Science.gov (United States)

    Maderia, Vitor M. C.; Pires, Euclides M. V.

    1986-01-01

    Discusses the value of electrophoresis in the fields of protein chemistry and biochemistry. Describes how to build an inexpensive electrophoresis setup for use in either research or teaching activities. Details the construction of both the separating device and the power supply. (TW)

  16. Proteomic analysis of the mouse brain following protein enrichment by preparative electrophoresis.

    Science.gov (United States)

    Xixi, Elena; Dimitraki, Ploumisti; Vougas, Kostantinos; Kossida, Sofia; Lubec, Gert; Fountoulakis, Michael

    2006-04-01

    Proteomics is a powerful technology to study the identity and levels of brain proteins. Changes of protein levels as well as modifications that occur in neurological disorders may be informative for the pathogenesis of these disorders and could result in the identification of potential drug targets and disease markers. To increase the capability of characterizing complex protein profiles, protein mixtures should be separated into simpler fractions, thus increasing the likelihood of detecting low-abundance proteins. Considering that low-abundance proteins are thought to be involved in important biological processes, identification of those low-copy-number gene products appears to be a scientific challenge. In the present study, proteomic analysis of adult mouse brain tissue was performed following enrichment by preparative electrophoresis. This was performed using the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Samples were electrophoresed in a cylindrical polyacrylamide gel and the proteins of the fractions collected were first analyzed by 1-D and then by 2-DE. Protein identification was performed by MALDI-TOF-MS. The present analysis resulted in the identification of 360 different gene products. Among those were transport proteins, transcription activators, signal transduction molecules as well as proteins with a number of other functions. Preparative electrophoresis is an efficient method for the enrichment of proteins of low molecular mass and may be useful in the investigation of disorders of the central nervous system.

  17. Isolation of Oat (Avena sativa L.) Total Proteins and Their Prolamin Fractions for 2D Electrophoresis.

    Science.gov (United States)

    Nałęcz, Dorota; Dziuba, Marta; Szerszunowicz, Iwona

    2017-01-01

    Appropriate sample preparation is essential to obtaining good results of two-dimensional gel electrophoresis (2-DE). For various reasons (particularly phenolic compounds, proteolytic enzymes, and cell-wall mucilages) the extraction of proteins from plant material, among them oat proteins, is difficult. During isolation all soluble substances that may interfere with the analysis (especially isoelectric focusing) are removed, and proteins of interest are separated from the remains. However, the applied procedure of isolation cannot be too extensive, because additional stages cause loss of the proteins.In this chapter, we describe a simple procedure for the isolation of oat total proteins and their prolamin fractions prior to 2-DE, without necessity of considerable purification. It can be used for oat protein fractionation, measurement of oat protein concentration, and their 2-DE analysis, with particular reference to prolamin fractions. The presented routine includes modified methods of plant seed proteins extraction and sequential Osborne extraction, based on oat protein solubility differences.

  18. Polyacrylamide gel electrophoresis-SDS as a tool to study myofibrillar proteins. A review.

    Directory of Open Access Journals (Sweden)

    Perez-Chabela, M. Lourdes

    2015-12-01

    Full Text Available Miofibrillar proteins are part of land and sea animals’ muscle. Nonetheless, even when muscle proteins are the same type of proteins, their structure, rigor mortis time, and biochemical process associated to muscle to meat conversion, are different among animal species. This review has the aim to describe the advantages of SDS-polyacrylamide gel electrophoresis (SDS-PAGE in the study of myofibrillar proteins structure, besides the influence of many parameters on this technique to obtain an electrophoretic profile. Applications of this technique as a diagnostic tool in the food science, ecology and health are described as well.

  19. Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.

    Science.gov (United States)

    Raghupathy, V; Oommen, Anna; Ramachandran, Anup

    2014-06-15

    Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. 2D gel electrophoresis and mass spectrometry identification and analysis of proteins.

    Science.gov (United States)

    Meleady, Paula

    2011-01-01

    Analysis of the protein expression patterns in clinical samples and cells by proteomic technologies offers opportunities to discover potentially new biomarkers for early detection and diagnosis of disease. One of the most widely used techniques to study the proteome of a biological system is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In particular, a modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labelling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE. In this chapter, we will introduce methods for the analysis of whole cell lysates from human cancer cell lines using 2D-DIGE and identification of differentially expressed proteins using liquid chromatography mass spectrometry, i.e. LC-MS/MS.

  1. Geometry-induced injection dispersion in single-cell protein electrophoresis.

    Science.gov (United States)

    Pan, Qiong; Herr, Amy E

    2018-02-13

    Arrays of microwells are widely used to isolate individual cells, facilitate high throughput cytometry assays, and ensure compatibility of those assays with whole-cell imaging. Microwell geometries have recently been utilized for handling and preparation of single-cell lysate, prior to single-cell protein electrophoresis. It is in the context of single-cell electrophoresis that we investigate the interplay of microwell geometry (circular, rectangular, triangular) and transport (diffusion, electromigration) on the subsequent performance of single-cell polyacrylamide gel electrophoresis (PAGE) for protein targets. We define and measure injector-induced dispersion during PAGE, and develop a numerical model of band broadening sources, experimentally validate the numerical model, and then identify operating conditions (characterized through the Peclet number, Pe) that lead to microwell-geometry induced losses in separation performance. With analysis of mammalian cells as a case study, we sought to understand at what Pe is the PAGE separation performance adversely sensitized to the microwell geometry. In developing design rules, we find that for the microwell geometries that are the most suitable for isolation of mammalian cells and moderate mass protein targets, the Pe is usually small enough (Pe geometry on protein PAGE of single-cell lysate. In extreme cases where the largest mammalian cells are analyzed (Pe > ∼20), consideration of Pe suggests using a rectangular - and not the widely used circular - microwell geometry to maximize protein PAGE separation performance. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.

    Science.gov (United States)

    Ma, Renli; McLeod, Cameron W; Tomlinson, Kerry; Poole, Robert K

    2004-08-01

    The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. Copyright 2004 Wiley-VCH Verlag GmbH and Co.

  3. Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts

    Science.gov (United States)

    Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter

    2010-04-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose-(concentration)dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 h post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

  4. Free flow electrophoresis separation and AMS quantitation of {sup 14}C-naphthalene-protein adducts

    Energy Technology Data Exchange (ETDEWEB)

    Buchholz, Bruce A., E-mail: bbuchholz@llnl.go [Center for AMS, LLNL, 7000 East Avenue, Livermore, CA 94551 (United States); Haack, Kurt W.; Sporty, Jennifer L. [Center for AMS, LLNL, 7000 East Avenue, Livermore, CA 94551 (United States); Buckpitt, Alan R.; Morin, Dexter [Department of Molecular Biosciences, School of Veterinary Medicine, UC Davis, Davis, CA 95616 (United States)

    2010-04-15

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose-(concentration)dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 muCi) of {sup 14}C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 h post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with {sup 14}C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

  5. Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Pramod Kumar Singh

    2016-01-01

    Full Text Available Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE across a broad range 3.0–10.0 immobilized pH gradient (IPG strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected in these accessions. In-gel protein expression patterns revealed three protein spots as upregulated and three other as downregulated. Using trypsin in-gel digestion, these differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS which showed 45% amino acid homology of chickpea seed storage proteins with Arabidopsis thaliana.

  6. Application of fluorescence difference gel electrophoresis technology in searching for protein biomarkers in chick myopia.

    Science.gov (United States)

    Lam, Thomas C; Li, King-Kit; Lo, Samuel C L; Guggenheim, Jeremy A; To, Chi Ho

    2007-11-01

    The lens-induced myopia (LIM) in response to concave lens (negative lens) is a well established animal model for studying myopia development. However, the exact visual and neurochemical signaling mechanisms involving myopic eye growth are yet to be elucidated. The feasibility of applying a novel two-dimensional fluorescence difference gel electrophoresis technique for global protein profilings and a search for differential protein expressions in LIM were explored in the present study. Two-dimensional polyacrylamide gel electrophoresis was performed employing a "minimal Lysine labeling" approach and a reverse CyeDye experimental protocol using retinal tissue from chicks. The retinal protein profiles between myopic and control eyes were found to be very similar. More than a thousand protein spots could be detected on a 2D gel. Sixteen and ten protein spots were found to be up-regulated and down-regulated respectively in the myopic eyes according to our preset criteria with the inclusion of an internal pool standard. About 65% of those filtered spots could be successfully identified by peptide mass fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry . Most of the differentially expressed proteins were found to be related to cytoskeletal or oxidative functions. According to the prediction of subcellular locations, most of them (about 84%) were classified as cytoplasmic proteins. The cellular functions for those differentially expressed proteins were reported and their possible involvements in the compensated eye growth were discussed. We have optimized a workable protocol for the study of the differential retinal protein expressions in the LIM using 2D-DIGE approach which was shown to have a number of advantages over the traditional 2D electrophoresis technique.

  7. Identification of phagocytosis-associated surface proteins of macrophages by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Howard, F D; Petty, H R; McConnell, H M

    1982-02-01

    Two-dimensional PAGE (P. Z. O'Farrell, H. M. Goodman, and P. H. O'Farrell. 1977. Cell. 12:1133-1142) has been employed to assess the effects of antibody-dependent phagocytosis on the cell surface protein composition of RAW264 macrophages. Unilamellar phospholipid vesicles containing 1% dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-cap-PE) were used as the target particle. Macrophages were exposed to anti-DNP antibody alone, vesicles alone, or vesicles in the presence of antibody for 1 h at 37 degrees C. Cell surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination at 4 degrees C. After detergent solubilization, membrane proteins were analyzed by two-dimensional gel electrophoresis. The resulting pattern of spots was compared to that of standard proteins. We have identified several surface proteins, not apparently associated with the phagocytic process, which are present either in a multichain structure or in several discretely charged forms. After phagocytosis, we have observed the appearance of two proteins of 45 and 50 kdaltons in nonreducing gels. In addition, we have noted the disappearance of a 140-kdalton protein in gels run under reducing conditions. These alterations would not be detected in the conventional one-dimensional gel electrophoresis. This evidence shows that phagocytosis leads to a modification of cell surface protein composition. Our results support the concept of specific enrichment and depletion of membrane components during antibody-dependent phagocytosis.

  8. Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L. Proteins and Protein Fractionations

    Directory of Open Access Journals (Sweden)

    Xiaoying Mao

    2014-01-01

    Full Text Available As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

  9. 2d electrophoresis of bovine milk proteins and milk fermented drink

    OpenAIRE

    Damir Mogut; Anna Iwaniak; Monika Hrynkiewicz; Jerzy Dziuba

    2015-01-01

    The aim of the study was the analysis of milk and milk fermented drink proteomes with the use of 2D electrophoresis. The criteria of proteins separation were the values of their isoelectric points (pI) and molecular weights (MW). Our results showed that milk and milk fermented drink proteomes consisted of 118 and 121 spots, respectively. The computer analysis revealed the identity of 95 spots in both proteomes. Non-identical spots indicated the changes resulting from the action of bact...

  10. An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

    Science.gov (United States)

    Tan, Niu J; Daim, Leona D J; Jamil, Amilia A M; Mohtarrudin, Norhafizah; Thilakavathy, Karuppiah

    2017-03-01

    Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Application of native agarose gel electrophoresis of serum proteins in veterinary diagnostics

    Directory of Open Access Journals (Sweden)

    Jania Bartosz

    2016-12-01

    Full Text Available Electrophoretic techniques, used to separate mixtures of electrically charged particles, are widely used in science. One of these techniques, native protein electrophoresis in an agarose gel, is applied in human and veterinary medicine. Changes in the proportions of individual protein fractions correspond to significant changes in the physiology of the body. Although the pattern obtained by electrophoretic separation rarely indicates a specific disease, it provides valuable information for the differential diagnosis. Decades of research on the types of patterns obtained in the case of particular diseases have led to the accumulation of substantial knowledge. The paper presents the available information on this topic. Serum protein electrophoresis is recommended in cases of increased levels of total protein in order to reveal the nature of the process. The basic information which can be obtained from electrophoretic separation includes the immune status of the organism. Both increased antigenic stimulation and immunodeficiency are clearly visible in electropherograms. Moreover, the level of heterogeneity of the corresponding protein fractions can help to distinguish between infectious diseases and cancer - multiple myeloma - the latter producing a homogeneous immunoglobulin fraction. Analysis of other protein fractions helps to detect or confirm an ongoing inflammatory process and provides information regarding liver function. Even when the concentration of total protein is within the reference range, this analysis can be recommended as a basic laboratory test.

  12. Completely monodisperse, highly repetitive proteins for bioconjugate capillary electrophoresis: development and characterization.

    Science.gov (United States)

    Lin, Jennifer S; Albrecht, Jennifer Coyne; Meagher, Robert J; Wang, Xiaoxiao; Barron, Annelise E

    2011-06-13

    Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.

  13. Urine protein electrophoresis study in dogs with pituitary dependent hyperadrenocorticism during therapy with trilostane

    Directory of Open Access Journals (Sweden)

    Douglas S. Caragelasco

    Full Text Available ABSTRACT: Hyperadrenocorticism is one of the most common endocrine disorders in dogs. Regarding to the kidneys, chronic hypercortisolemia can cause damage to the glomerulus, and evolve into chronic kidney disease. This study evaluated nine normotensive dogs with pituitary dependent hyperadrenocorticism, before and after therapy with trilostane, during the follow-up period of six months, in order to investigate the development of pathological proteinuria by quantitative (urinary protein-to-creatinine ratio and qualitative (urinary protein electrophoresis methods, and also to monitor its intensity over the course of the disease and therapy. The main renal lesion detected in dogs with hyperadrenocorticism was in the tubular segment, evidenced by the prevalence of urinary protein bands of lower molecular weight, indicating the lack absorption of these proteins in the proximal segment of the nephron. Low molecular weight proteins persisted throughout the follow-up. Regarding the future of routine veterinary medical clinic in the care of patients with hyperadrenocorticism, the assessments of proteinuria determinations by the urinary protein-to-creatinin ratio and urinary protein electrophoresis, according to the results obtained in this study, can add more information about the renal damage in these animals, and contribute to the prognosis.

  14. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Jae Eun Lee

    2015-06-01

    Full Text Available Two dimensional-fluorescence difference gel electrophoresis (2D DIGE is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

  15. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Science.gov (United States)

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

  16. Use of electrophoresis and immunoelectrophoresis in taxonomic and pollution studies

    Digital Repository Service at National Institute of Oceanography (India)

    Menezes, M.R.; Qasim, S.Z.

    Studies were conducted on the electrophoresis of blood serum and eye lens proteins of 5 fishes and immunoelectrophoresis of the soluble lens proteins of 10 fishes. The effects of a toxic pollutant (mercury) on the electrophoretic patterns...

  17. Electrophoresis-tutor: an image-based personal computer program that teaches clinical interpretation of protein electrophoresis patterns of serum, urine, and cerebrospinal fluid.

    Science.gov (United States)

    Astion, M L; Rank, J; Wener, M H; Torvik, P; Schneider, J B; Killingsworth, L M

    1995-09-01

    High-resolution protein electrophoresis of serum, urine, and cerebrospinal fluid (CSF) can aid in the diagnosis of multiple myeloma, amyloidosis, macroglobulinemia, multiple sclerosis, and other diseases. Electrophoresis-Tutor is a personal computer program based on approximately 150 digital images that teaches the clinical interpretation of agarose gel electrophoretic patterns. The program is divided into the following sections: introduction, CSF, serum, urine, review of disease states, program navigator, and final exam. The CSF section describes normal and abnormal CSF findings with emphasis on oligoclonal banding, as seen in the CSF of patients with multiple sclerosis. The serum section emphasizes monoclonal gammopathy patterns but also has detailed descriptions of inflammation, liver disease, protein-losing disorders, genetic deficiencies, and other patterns. Monoclonal gammopathy is described in the context of specific associated clinical conditions (e.g., myeloma, amyloidosis). For each monoclonal gammopathy example, results of standard electrophoresis, densitometry, and immunofixation are presented. The review of disease states uses animation to illustrate the development and remission of a variety of pathological patterns. The program navigator allows the user to jump quickly to any place in the program. The optional exam contains 20 questions, and detailed feedback is given after each question. Electrophoresis-Tutor can be used as a stand-alone teaching tool, a companion to traditional instruction, or a reference source.

  18. Parathyroid hormone-related protein blood test

    Science.gov (United States)

    ... ency/article/003691.htm Parathyroid hormone-related protein blood test To use the sharing features on this page, ... measures the level of a hormone in the blood, called parathyroid hormone-related protein. How the Test is Performed A blood sample is needed . How ...

  19. Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

    Directory of Open Access Journals (Sweden)

    J Vejayan

    2010-01-01

    Full Text Available Snake venoms comprise a highly complex mixture of proteins, which requires for their characterization the use of versatile two-dimensional electrophoresis techniques. In the present study, venoms obtained from eight snakes (Ophiophagus hannah, Naja kaouthia, Naja sumatrana, Bungarus fasciatus, Trimeresurus sumatranus, Tropidolaemus wagleri, Enhydrina schistosa and Calloselasma rhodostoma commonly found in Malaysia were separated based on two independent properties, isoelectric point (pI and molecular weight (MW. Many differences in snake venoms at the inter-family, inter-subfamily, inter-genus and inter-species levels were revealed. Notably, proteins from individuals of the Viperidae family - Trimeresurus sumatranus, Tropidolaemus wagleri and Calloselasma rhodostoma - were found to be numerous and scattered by the two-dimensional gel electrophoresis (2DE specifically in regions between 37 and 100 kDa compared to the Elapidae venom proteins. The latter were clustered at the basic and lower molecular mass region (less than 20 kDa. Trains of spots were commonly observed, indicating that these proteins may be derived from post-translational modifications. Ophiophagus hannah (Elapidae revealed a great amount of protein spots in the higher molecular mass range when compared to Enhydrina schistosa, Naja kaouthia, Naja sumatrana and Bungarus fasciatus. Overall 2DE showed large differences in the venom profile of each species, which might be employed as an ancillary tool to the identification of venomous snake species.

  20. Leaf Protein Electrophoresis and Taxonomy of Species of Jatropha L. (Euphorbiaceae

    Directory of Open Access Journals (Sweden)

    Olaniran Temitope OLADIPO

    2012-08-01

    Full Text Available The systematic relationship existing among members of the all important genus Jatropha was studied using leaf protein electrophoresis. The aim was to identify possible taxonomic importance of the protein profile in the estimation and elucidation of the taxonomic affinity of the six species of Jatropha (Jatropha curcas Linn., J. podagrica Hook., J. gossypifolia Linn., J. mutifida Linn., J. tanjorensis Ellis & Saroja and J. integerrima Linn. found in Nigeria. The species were screened for total protein banding patterns using gel electrophoresis. Young leaves (0.8 g of the plants were washed with distilled water and macerated with sterile mortar and pestle in 0.8% Phosphate Buffer-Saline (PBS containing 0.4 M NaCl at pH 8.0. Results reveal that protein banding pattern was taxon specific. Generic band occurs at 8.3. The highest number of interspecific bands (4 exists between J. podagrica and J. multifida. Variations exist not only in the number of bands but also in the intensity of the bands. Sokal and Sneath coefficient of similarity ranges between 11.1-44.4 %. Single linkage Cluster Analysis (SLCA of the relative mobility values of the protein in the taxa shows partial agreement with current sub generic and sectional delimitation of the species based on morphology and anatomy of the species.

  1. Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection

    Directory of Open Access Journals (Sweden)

    Angelo Zinellu

    2017-01-01

    Full Text Available Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and reproducible capillary electrophoresis method with UV detection for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolysed samples were dried and resuspended with water and directly injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4 : 1, v/v allowed full analyte identification within 11 min. Precision tests indicated an elevated reproducibility with an interassay CV of 1.98% when starting from 2 μg of the extracted DNA. The method was successfully tested by measuring the DNA methylation degree both in healthy volunteers and in reference calf thymus DNA.

  2. Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis

    Directory of Open Access Journals (Sweden)

    Lin Qiu

    2014-01-01

    Full Text Available In this report, fluorescence detection coupled capillary electrophoresis (CE-FL was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

  3. Rapid Seafood Species Identification Using Chip-Based Capillary Electrophoresis and Protein Pattern Matching.

    Science.gov (United States)

    Walker, Calvin C; Lassitter, Cheryl L; Lynn, Shannara N; Ford, Courtney B; Rademacher, Kevin R; Ruple, Angela D; Bell, Jon W

    2017-09-01

    Authenticity is crucial to the seafood industry, as substitution and mislabeling have important economic, environmental, and food safety consequences. To address this problem, protein profiling and software algorithm techniques were developed to classify fish muscle samples by species. The method uses water-based protein extraction, chip-based microfluidic electrophoresis (Agilent 2100 Bioanalyzer) for the analysis of high abundance fish muscle proteins, and a novel data analysis method for species-specific protein pattern recognition. The method's performance in distinguishing commercially important fish from commonly reported substitutions was evaluated using sensitivity, specificity, and accuracy determinations with all three performance measures at >98% for common substitutions. This study demonstrates that uncooked seafood products of commercially important species of catfish, snapper, and grouper can be rapidly distinguished from commonly substituted species with a high level of confidence. A tiered testing approach to seafood species verification by sequentially applying a rapid screening method and DNA testing is proposed to more effectively ensure accurate product labeling.

  4. An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins.

    Science.gov (United States)

    Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

    2017-01-01

    The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for

  5. Characterization of Bacillus strains and hoax agents by protein profiling using automated microfluidic capillary electrophoresis.

    Science.gov (United States)

    McLaughlin, Jessica; Nelson, Michelle; McNevin, Dennis; Roffey, Paul; Gahan, Michelle E

    2014-09-01

    In recent times, but especially since 2001, bioterrorism has been of increasing concern. In addition to the use of biological agents, including Bacillus anthracis (anthrax), there have been numerous hoax white powder "scares." It is imperative to rapidly and accurately identify any suspicious powder as hazardous or hoax. Classical methods for identification typically rely on time-consuming cultivation or highly specific molecular tests which are limited if the agent is unknown. Faster and field portable methods for analysis of suspicious powders are urgently required. Potential hoax agents, including Bacillus species and household powders, were analyzed using automated microfluidic capillary electrophoresis to determine if protein profiling can distinguish between, and identify, samples. Distinctive protein profiles were produced for Bacillus species, with the presence and/or absence of certain bands, aiding identification. In particular B. anthracis Sterne strain contained a distinctive doublet band above 100 kDa which was not present in any other Bacillus species or hoax agents examined. The majority of powders produced distinctive banding that could enable the identification of the sample while simultaneously ruling out B. anthracis with a high degree of confidence. Results show automated microfluidic capillary electrophoresis can rapidly and reproducibly characterize Bacillus species and hoax agents based on protein profiles without the need for culture. Results were reproducible and there was enhanced resolution and rapidity compared to traditional protein profiling methods. Results show this technique is amenable to field use at a bioterrorism incident, thereby providing essential information to investigators regarding containment and treatment strategies.

  6. Serum biochemistry and native protein electrophoresis in diarrheic calves with arthritis

    Directory of Open Access Journals (Sweden)

    Pekcan M.

    2012-01-01

    Full Text Available In this study, serum biochemistry and native protein electrophoresis in newborn calves with diarrhea and arthritis, were performed in order to evaluate the changes along with clinical findings for their possible application in the diagnosis and prognosis of disease. Based on clinical examination, animals were allotied into two groups comprising either diseased or healthy animals. Urea, creatinine, ALT, AST, LDH, albumin, total protein, glucose, total cholesterol, uric acid and iron levels were determined in the sera. Serum protein native polyacrilamide gel electrophoresis (nPAGE was performed followed by protein band ratio estimation supported with densitometry at 596 nm. Differences between the average mean of healthy and diseased animals were compared statistically (Kruskal-Walley test. In this study a decrease in serum glucose and cholesterol values (p<0.001, increase in urea, LDH levels and α1-and α2-globulin levels (p<0.01 and p<0.05 respectively were found to be associated with the disease. As a result, the observed significant changes in biochemical parameters and clinical investigation in calves, suggesting acute inflammation causing the decrease in glucose and increase in α-globulins, may be of prognostic value.

  7. Re-use of commercial microfluidics chips for DNA, RNA, and protein electrophoresis.

    Science.gov (United States)

    Nguyen, Thi; Kwak, Sukyoung; Karpowicz, Steven J

    2014-11-01

    Microfluidics chip technology is a powerful and convenient alternative to agarose gels and PAGE, but costs can be high due to certain chips being non-reusable. Here we describe a method to regenerate, re-use, and store Agilent DNA, RNA, and protein electrophoresis chips designed for use in the Bioanalyzer 2100. By washing the sample wells and displacing the old gel matrix with new gel-dye mix, we have run samples on the same chip up to ten times with negligible loss of signal quality. Chips whose wells were loaded with buffer or water were stored successfully for one week before re-use.

  8. Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Horká, Marie; Tesařová, Marie; Karásek, Pavel; Růžička, F.; Holá, V.; Sittová, M.; Roth, Michal

    2015-01-01

    Roč. 868, APR (2015), s. 67-72 ISSN 0003-2670 R&D Projects: GA ČR(CZ) GAP106/12/0522; GA MV VG20102015023 Institutional support: RVO:68081715 Keywords : capillary zone electrophoresis * Staphylococcus aureus * human whole blood Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.712, year: 2015 http://hdl.handle.net/11104/0245801

  9. Protein and cholesterol electrophoresis of plasma samples from captive cownose ray (Rhinoptera bonasus).

    Science.gov (United States)

    Cray, Carolyn; Rodriguez, Marilyn; Field, Cara; McDermott, Alexa; Leppert, Lynda; Clauss, Tonya; Bossart, Gregory D

    2015-11-01

    Our study was undertaken to assess the application of semiautomated methods available at the reference laboratory level for the evaluation of plasma protein and cholesterol via electrophoresis in samples from cownose rays (Rhinoptera bonasus). Three groups of animals were assessed: clinically normal, clinically abnormal, and parasitized with leeches. As reported previously, the albumin band was negligible; the protein electrophoretograms were dominated by a large beta-globulin fraction. While the group of samples from the leech-parasitized rays did not show any large differences, the abnormal group exhibited significantly elevated total solids and cholesterol levels. The latter was related to a significant increase in very low density lipoprotein levels. The results demonstrate the potential application of these laboratory methods in quantitation of plasma proteins and cholesterol fractions in subclass Elasmobranchii. © 2015 The Author(s).

  10. REVIEW: The Early Application of Electrophoresis of Protein in Higher Plant Taxonomy

    Directory of Open Access Journals (Sweden)

    SURANTO

    2002-07-01

    Full Text Available The aims of this research are firstly, to study the advantages of electrophoretic techniques. Secondly, to look at the usefulness of a few mediums support of electrophoretic proteins especially the acrylamide gel. Thirdly, to examine the number of plant organs which could be used as the sources of plant proteins, and how these plants protein should be applied in the medium support that has been selected. Besides, the staining and detection procedures would be described, while the application of electrophoretic approach in higher plant taxonomy will also be evaluated. In this study we recorded that a number of taxonomic problems usually caused by morphological complexity within species can be solved using this experimental approach of electrophoresis. This method has been considered very useful in helping taxonomists making decisions.

  11. Multivariate data analysis of two-dimensional gel electrophoresis protein patterns from few samples

    DEFF Research Database (Denmark)

    Jensen, Kristina Nedenskov; Jessen, Flemming; Jørgensen, Bo

    2008-01-01

    on single spot differences, but on the covariance structure between proteins. However, their outcome is dependent on data scaling, and they may fail in producing valid multivariate models due to the much higher number of "irrelevant" spots present in the gels. The case where only few gels are available......One application of 2D gel electrophoresis is to reveal differences in protein pattern between two or more groups of individuals, attributable to their group membership. Multivariate data analytical methods are useful in pinpointing the spots relevant for discrimination by focusing not only...... and where the aim is to find as many as possible of the group-dependent proteins seems particularly difficult to handle. The present paper investigates such a case regarding the effect of scaling and of prefiltering by univariate nonparametric statistics on the selection of spots. Besides, a modified...

  12. Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS- PAGE) of Irradiated Wheat Flour Proteins

    International Nuclear Information System (INIS)

    Souzan, R.M.

    1999-01-01

    Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) of wheat (Triticum aestivum L) flour have revealed 23 polypeptides of molecular weights between 170 and 11.57 KDa, High molecular weight glutenin subunits (LMW-GS) were distinguished. Densitometric analysis of the gel showed the effect of radiation on polypeptide constitution at radiation energy up to 7.5 kGy. Irradiation of wheat flour with 2.5 kGy have resulted in a slight increase in the molecular weight of wheat flour protein subunits. The increase of irradiation dose to 5.0 kGy has also induced an additional increase of molecular weight of protein subunits. The continuity in application of more radiation energy to a level of 7.5 kGy have resulted in the prevalence of degradation processes of all protein subunits more than the aggregation

  13. [Analysis of total proteins in the seed of almond (Prunus dulcis) by two-dimensional electrophoresis].

    Science.gov (United States)

    Li, Dong-dong; He, Shao-heng

    2004-07-01

    To analyse the total proteins in the seeds of almond (Prunus dulcis), one of the popular ingestent allergens in China, by two-dimensional electrophoresis. The total proteins of the seeds were extracted by trichloracetic acid (TCA) method, and then separated by isoelectric focusing as first dimension and SDS-PAGE as the second dimension. The spots of proteins were visualized by staining with Coomassie Brilliant Blue R-250. After analysis with software (ImageMaster 2D), 188 different proteins were detected. The isoelectric points (pI) for approximately 28% of total proteins were between 4.5-5.5, and the relative molecular mass (M(r)) of approximately 62% total proteins were between (20-25)x10(3). This was the first high-resolution, two-dimensional protein map of the seed of almond (Prunus dulcis) in China. Our finding has laid a solid foundation for further identification, characterization, gene cloning and standardization of allergenic proteins in the seed of almond (Prunus dulcis).

  14. Automated SDS Depletion for Mass Spectrometry of Intact Membrane Proteins though Transmembrane Electrophoresis.

    Science.gov (United States)

    Kachuk, Carolyn; Faulkner, Melissa; Liu, Fang; Doucette, Alan A

    2016-08-05

    Membrane proteins are underrepresented in proteome analysis platforms because of their hydrophobic character, contributing to decreased solubility. Sodium dodecyl sulfate is a favored denaturant in proteomic workflows, facilitating cell lysis and protein dissolution; however, SDS impedes MS detection and therefore must be removed prior to analysis. Although strategies exist for SDS removal, they provide low recovery, purity, or reproducibility. Here we present a simple automated device, termed transmembrane electrophoresis (TME), incorporating the principles of membrane filtration, but with an applied electric current to ensure near-complete (99.9%) removal of the surfactant, including protein-bound SDS. Intact proteins are recovered in solution phase in high yield (90-100%) within 1 h of operation. The strategy is applied to protein standards and proteome mixtures, including an enriched membrane fraction from E. coli, resulting in quality MS spectra free of SDS adducts. The TME platform is applicable to both bottom-up MS/MS as well as LC-ESI-MS analysis of intact proteins. SDS-depleted fractions reveal a similar number of protein identifications (285) compared wit a non-SDS control (280), being highly correlated in terms of protein spectral counts. This fully automated approach to SDS removal presents a viable tool for proteome sample processing ahead of MS analysis. Data are available via ProteomeXchange, identifier PXD003941.

  15. Quantitative analysis of protein phosphorylation using two-dimensional difference gel electrophoresis.

    Science.gov (United States)

    Deng, Zhiping; Bu, Shuolei; Wang, Zhi-Yong

    2012-01-01

    Posttranslational modifications of proteins, especially phosphorylation and dephosphorylation, play an important role in signal transduction and cellular regulation in plants. Both 2-DE gel-based and non-gel-based proteomic technologies can monitor the changes in phosphorylation state of proteins. In this chapter, we describe two protocols for discovery and validation of differential protein phosphorylation using affinity enrichment of phosphoproteins by immobilized metal affinity chromatography (IMAC) or protein immunoprecipitation (IP) followed by two-dimensional difference gel electrophoresis (2-D DIGE). We name these methods IMAC-DIGE and IP-DIGE. For IMAC-DIGE, phosphoproteins are enriched from tissue extract using GaCl(3)-based IMAC and then analyzed by 2-D DIGE, which reveals changes of protein phosphorylation as protein spot shifts. IMAC enrichment improves detection of low-abundance regulatory phosphoproteins. For IP-DIGE, proteins of interest can be immunopurified and then analyzed by 2-D DIGE to confirm changes of posttranslational modifications that alter the charge or size of the proteins.

  16. Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating.

    Science.gov (United States)

    Unterlander, Nicole; Doucette, Alan Austin

    2018-02-08

    SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass-based separations (e.g. SDS-PAGE, GELFrEE). However, SDS interferes with mass spectrometry and so it must be removed prior to analysis. We recently introduced an electrophoretic platform, termed transmembrane electrophoresis (TME), enabling extensive depletion of SDS from proteins in solution with exceptional protein yields. However, our prior TME runs required 1 h to complete, being limited by Joule heating which causes protein aggregation at higher operating currents. Here, we demonstrate effective strategies to maintain lower TME sample temperatures, permitting accelerated SDS depletion. Among these strategies, the use of a magnetic stir bar to continuously agitate a model protein system (BSA) allows SDS to be depleted below 100 ppm (>98% removal) within 10 min of TME operations, while maintaining exceptional protein recovery (>95%). Moreover, these modifications allow TME to operate without any user intervention, improving throughput and robustness of the approach. Through fits of our time-course SDS depletion curves to an exponential model, we calculate SDS depletion half-lives as low as 1.2 min. This promising electrophoretic platform should provide proteomics researchers with an effective purification strategy to enable MS characterization of SDS-containing proteins. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

    Science.gov (United States)

    Burré, Jacqueline; Beckhaus, Tobias; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

    2006-09-01

    Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

  18. Serum globulin electrophoresis

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003544.htm Serum globulin electrophoresis To use the sharing features on this page, please enable JavaScript. The serum globulin electrophoresis test measures the levels of proteins called globulins ...

  19. Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting

    DEFF Research Database (Denmark)

    Andersen, H; Birkelund, Svend; Christiansen, Gunna

    1987-01-01

    The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others...

  20. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    Directory of Open Access Journals (Sweden)

    A. Kubicz

    2015-01-01

    Full Text Available Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.. The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance of these bands. RNase activity was detected in 4 main zones, in some of them additional subbands were visible. Differences between the three examined varieties were reflected in the occurence of the particular activity zones or their subbands.

  1. Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis.

    Science.gov (United States)

    Poitevin, Martine; Hammad, Karim; Ayed, Ichraf; Righetti, Pier Giorgio; Peltre, Gabriel; Descroix, Stephanie

    2008-08-01

    The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.

  2. Analyzing import intermediates of mitochondrial proteins by blue native gel electrophoresis.

    Science.gov (United States)

    Waizenegger, Thomas; Rapaport, Doron

    2007-01-01

    Blue native gel electrophoresis (BNGE) is a powerful tool for analyzing native protein complexes from biological membranes as well as water-soluble proteins. It can be used for determining relative molecular masses of protein complexes and their subunit composition and for the detection of subcomplexes. We describe the analysis by BNGE of in vitro import reactions composed of radiolabeled precursor proteins and isolated mitochondria. Such an analysis is a powerful tool to follow import intermediates and to study assembly of protein complexes. Analysis of import reactions by BNGE provides information on the molecular mass of the complex with which the imported precursor is associated. In addition, components of such a complex can be identified by incubating the mitochondrial lysate with either soluble antibodies or antibodies coupled to protein A matrix. The binding of soluble antibodies to specific complexes results in an observed shift in their apparent molecular mass (antibody shift). Alternatively, addition of matrix-bound antibodies followed by removal of the matrix from the mixture will result in depletion of the specific complex from the mitochondrial lysate (antibody depletion). The experimental details of these techniques are described.

  3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE of urinary protein in acute kidney injury

    Directory of Open Access Journals (Sweden)

    Sufi M Suhail

    2011-01-01

    Full Text Available Recent experimental and clinical studies have shown the importance of urinary proteomics in acute kidney injury (AKI. We analyzed the protein in urine of patients with clinical AKI using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE for its diagnostic value, and followed them up for 40 months to evaluate prognosis. Urine from 31 consecutive cases of AKI was analyzed with SDS-PAGE to determine the low, middle and high molecular weight proteins. Fractional excretion of sodium (FENa was estimated from serum and urine creatinine and sodium (Na. The cases were followed-up for 40 months from the end of the recruitment of study cases. Glomerular protein was higher in the hematuria group when compared with the non-hematuria group (P <0.04 and in the AKI group than in the acute on chronic renal failure (AKI-on-CRF group (P <0.002. Tubular protein was higher in the AKI-on-CRF group (P <0.003 than in the AKI group. Tubular protein correlated with FENa in groups with diabetes mellitus (DM, AKI-on-CRF, and without hematuria (P <0.03, P <0.02 and P <0.004, respectively. Pattern of protein did not differ between groups with and without DM and clinical acute tubular necrosis (ATN. At the end of 40 months follow-up, category with predominantly glomerular protein progressed to chronic renal failure (CRF or end-stage renal failure in higher proportion (P <0.05. In clinical AKI, we observed that glomerular protein dominated in cases with glomerular insult, as indicated by hematuria. Tubular protein was common in the study cases with CRF, DM and cases without hematuria. This indicates tubulo-interstitial injury for AKI in these cases. Patients with predominantly glomerular protein had an adverse outcome.

  4. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    Energy Technology Data Exchange (ETDEWEB)

    Higa, A.M.; Noronha, M.D.N. [Universidade do Estado do Amazonas (UEA), Manaus, AM (Brazil). Rede Proteomica do Amazonas (Proteam). Lab. de Genomica e Proteomica; Rocha-Oliveira, F.; Lopez-Lozano, J.L.L. [Universidade Federal do Amazonas (UFAM), Manaus, AM (Brazil). Pos-Graduacao em Biotecnologia

    2008-07-01

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with {approx} 60, 70 and 80 kDa were detected in gel acidic region with pI {approx} 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI {approx} 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with {approx} 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI {approx} 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course.

  5. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    International Nuclear Information System (INIS)

    Higa, A.M.; Noronha, M.D.N.; Rocha-Oliveira, F.; Lopez-Lozano, J.L.L.

    2008-01-01

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with ∼ 60, 70 and 80 kDa were detected in gel acidic region with pI ∼ 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI ∼ 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with ∼ 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI ∼ 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course

  6. Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Parkhey, Suruchi; Chandrakar, Vibhuti; Naithani, S C; Keshavkant, S

    2015-10-01

    Protein extraction for two-dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one-dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77-95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Prediction of protein-DNA complex mobility in gel-free capillary electrophoresis.

    Science.gov (United States)

    Bao, Jiayin; Krylova, Svetlana M; Cherney, Leonid T; Hale, Robert L; Belyanskaya, Svetlana L; Chiu, Cynthia H; Arico-Muendel, Christopher C; Krylov, Sergey N

    2015-02-17

    Selection of protein binders from highly diverse combinatorial libraries of DNA-encoded small molecules is a highly promising approach for discovery of small-molecule drug leads. Methods of kinetic capillary electrophoresis provide the high efficiency of partitioning required for such selection but require the knowledge of electrophoretic mobility of the protein-ligand complex. Here we present a theoretical approach for an accurate estimate of the electrophoretic mobility of such complexes. The model is based on a theory of the thin double layer and corresponding expressions used for the mobilities of a rod-like short oligonucleotide and a sphere-like globular protein. The model uses empirical values of mobilities of free protein, free ligand, and electroosmotic flow. The model was tested with a streptavidin-dsDNA complex linked through biotin (small molecule). The deviation of the prediction from the experimental mobility did not exceed 4%, thus confirming that not only is the model adequate but it is also accurate. This model will facilitate reliable use of KCE methods for selection of drug leads from libraries of DNA-encoded small molecules.

  8. Muscle protein analysis. II. Two-dimensional electrophoresis of normal and diseased human skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Giometti, C.S. (Argonne National Lab., IL); Barany, M.; Danon, M.J.; Anderson, N.G.

    1980-07-01

    High-resolution two-dimensional electrophoresis was used to analyze the major proteins of normal and pathological human-muscle samples. The normal human-muscle pattern contains four myosin light chains: three that co-migrate with the myosin light chains from rabbit fast muscle (extensor digitorum longus), and one that co-migrates with the light chain 2 from rabbit slow muscle (soleus). Of seven Duchenne muscular dystrophy samples, four yielded patterns with decreased amounts of actin and myosin relative to normal muscle, while three samples gave patterns comparable to that for normal muscle. Six samples from patients with myotonic dystrophy also gave normal patterns. In nemaline rod myopathy, in contrast, the pattern was deficient in two of the fast-type myosin light chains.

  9. Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

    DEFF Research Database (Denmark)

    Radzikowski, Louise; Nesic, Ljiljana; Hansen, H.B.

    2002-01-01

    The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Ronhave, Denmark, were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2-D...... electrophoresis. The gels were scanned, compared using ImageMaster(R) software and the data sets were analyzed by principal component analysis (PCA) using THE UNSCRAMBLER software. Afterwards MATLAB was used to make a cluster analysis of the varieties based on PCA. The analysis of the gels showed...... separately. When the results were combined from the two years five varieties could be differentiated. The results from the PCA confirmed the finding of the unique spots and cluster analysis was made in order to illustrate the results. The combination of the results from 2-D electrophoresis and other grain...

  10. Streptomycin induced protein expression analysis in Mycobacterium tuberculosis by two-dimensional gel electrophoresis & mass spectrometry.

    Science.gov (United States)

    Sharma, Prashant; Kumar, Bhavnesh; Singhal, Neelja; Katoch, Vishwa Mohan; Venkatesan, Krishnamurthy; Chauhan, Devendra Singh; Bisht, Deepa

    2010-10-01

    The resistance of Mycobacterium tuberculosis to streptomycin, a core drug for treatment of category II tuberculosis (TB) has posed a major challenge to the health providers as well as research workers worldwide and has severely compromised the therapeutic options. A significant proportion of streptomycin resistant M. tuberculosis isolates failed to show mutations in conventional targets like rpsL and rrs. Although efflux, permeability, etc. are also known to contribute, yet a substantial proportion of isolates remains resistant suggesting involvement of other unknown mechanism. A resistant isolate may show altered gene as well as protein expression under drug induced conditions and a whole cell proteome analysis under induced conditions might help in further understanding the mechanisms of drug resistance. The present study was therefore designed with the objective to identify proteins related to streptomycin resistance in M. tuberculosis isolate grown in presence and absence of streptomycin (SM). A clinical isolate of M. tuberculosis from Mycobacterial Repository Centre at the Institute (NJIL & OMD), Agra was grown in Sauton's medium for 36 h with/without subinhibitory concentration of the drug (2 μg/ml) and the cell lysate of isolates was prepared by sonication and centrifugation. Two-dimensional (2D) gel electrophoresis was employed to study the protein profile. The selected proteins were finally identified by MALDI-TOF mass spectrometry. Our study revealed eight inducible proteins (DnaK, fabG4, DNA-binding, hypothetical, two 14 kDa antigen and two 10 kDa chaperonin) that were upregulated in the presence of drug. This preliminary study has thrown light on whether or not and how the resistant isolate responds to streptomycin at its non-toxic but sub-inhibitory concentration. An in-depth study of the upregulated proteins will give an insight into probable sites of drug action other than established primary sites.

  11. Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

    DEFF Research Database (Denmark)

    Radzikowski, Louise; Nesic, Ljiljana; Hansen, H.B.

    2002-01-01

    The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Ronhave, Denmark, were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2-D electrophor......The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Ronhave, Denmark, were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2-D...... electrophoresis. The gels were scanned, compared using ImageMaster(R) software and the data sets were analyzed by principal component analysis (PCA) using THE UNSCRAMBLER software. Afterwards MATLAB was used to make a cluster analysis of the varieties based on PCA. The analysis of the gels showed...... separately. When the results were combined from the two years five varieties could be differentiated. The results from the PCA confirmed the finding of the unique spots and cluster analysis was made in order to illustrate the results. The combination of the results from 2-D electrophoresis and other grain...

  12. [A case of multiple myeloma showing marked differences in serum IgG levels between protein electrophoresis and turbidimetry].

    Science.gov (United States)

    Shin, Jeong Won; Park, Rojin; Choi, Tae Youn

    2008-08-01

    We report a case of multiple myeloma showing marked differences in serum Immunoglobulin G (IgG) levels between serum protein electrophoresis and turbidimetry. A 47-yr old man was admitted to our hospital due to severe back pain and diagnosed as having IgG-kappa type multiple myeloma. Serum protein level was 14.4 g/dL at the time of diagnosis. Serum IgG level was 8.5 g/dL by serum protein electrophoresis, but 11.6 g/dL by turbidimetry. The patient's clinical conditions had improved after receiving VAD (vincristine, adriamycin, dexamethasone) and VTD (vincristine, thalidomide, dexamethasone) chemotherapy and there were no differences in IgG levels between electrophoresis and turbidimetry when serum IgG levels were less than 3.0 g/dL. According to this, we considered that both protein electrophoresis and turbidimetry should be needed to quantify serum immunoglobulins for diagnosis and follow-up of the patients with monoclonal gammopathy.

  13. Electrophoresis of the non-structural proteins from normal and atrophic muscles of the rabbit and of man

    NARCIS (Netherlands)

    Haan, A.M.F.H.

    1953-01-01

    The proteins of normal and atrophied skeletal muscles of the rabbit and of man, soluble in salt solutions of low ionic strength and pH about 7, were studied by means of electrophoresis. Atrophy of rabbit leg muscles was caused by severance of the motoric nerve, by immobilisation of a leg in a

  14. Rapid capillary coating by epoxy-poly-(dimethylacrylamide): Performance in capillary zone electrophoresis of protein and polystyrene carboxylate

    Czech Academy of Sciences Publication Activity Database

    Chiari, M.; Cretich, M.; Šťastná, Miroslava; Radko, S. P.; Chrambach, A.

    2001-01-01

    Roč. 22, č. 4 (2001), s. 656-659 ISSN 0173-0835 Institutional research plan: CEZ:AV0Z4031919 Keywords : capillary coating * capillary zone electrophoresis * proteins Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.282, year: 2001

  15. Electrophoresis in space at zero gravity

    Science.gov (United States)

    Bier, M.; Snyder, R. S.

    1974-01-01

    Early planning for manufacturing operations in space include the use of electrophoresis for purification and separation of biological materials. Greatly simplified electrophoresis apparatus have been flown in the Apollo 14 and 16 missions to test the possibility of stable liquid systems in orbit. Additionally, isoelectric focusing and isotachophoresis are of particular interest as they offer very high resolution and have self-sharpening boundaries. The value of possible space electrophoresis is substantial. For example, present technology permits large fractionation of only a few of blood proteins many fractions, and separated cell populations are needed for research.

  16. Direct analysis of formate in human plasma, serum and whole blood by in-line coupling of microdialysis to capillary electrophoresis for rapid diagnosis of methanol poisoning

    OpenAIRE

    Kubáň, P. (Pavel); Boček, P. (Petr)

    2013-01-01

    A microdialytic device was in-line coupled to capillary electrophoresis for direct injection of blood samples. Its performance was demonstrated on rapid analysis of formic acid in various blood samples including serum samples of a patient diagnosed with acute methanol poisoning.

  17. CYPRINIDS TOTAL BLOOD PROTEINS DETERMINATION

    Directory of Open Access Journals (Sweden)

    TANŢI PATRICHE

    2009-10-01

    Full Text Available In aquaculture to get a high production is conditioned by awareness and keeping of an unaltered health condition of the biological material. To be aware of the health condition of the biological material in a fish farm allows us to establish the preventive measures required to prevent spreading of a disease and the treatment to be applied in case that a mass disease occurs. The level of the total protein in serum is, first of all, a synthetically indicator of the nutritional condition of the organism, presenting, at the same time, ample qualitative and quantitative variations depending on species, age, sex, stage of sexual maturity, water temperature and especially in correlation with the health condition of fish. Modification in value of the total protein point out some metabolic perturbations in fish body.

  18. Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of (/sup 35/S)methionine-labeled proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tabaqchali, S.; O' Farrell, S.; Holland, D.; Silman, R.

    1986-01-01

    A typing method for Clostridium difficile based on the incorporation of (/sup 35/S)methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. difficile.

  19. Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Lundemose, AG; Birkelund, Svend; Larsen, PM

    1990-01-01

    The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis. By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after...... infection. The early proteins were synthesized throughout the 30-h period investigated, but the synthesis of three proteins of 75, 62, and 45 kilodaltons decreased from 26 to 30 h postinfection. Pulse-chase analysis showed that the signals from the same three proteins declined 26 to 30 h after infection....... Three of the early proteins were identified as the S1 ribosomal protein, the GroEL-like protein, and DnaK-like protein, respectively....

  20. High-throughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation.

    Science.gov (United States)

    Allmendinger, Andrea; Dieu, Le-Ha; Fischer, Stefan; Mueller, Robert; Mahler, Hanns-Christian; Huwyler, Jörg

    2014-10-01

    Viscosity characterization of protein formulations is of utmost importance for the development of subcutaneously administered formulations. However, viscosity determinations are time-consuming and require large sample volumes in the range of hundreds of microliters to a few milliliters, depending on the method used. In this article, an automated, high-throughput method is described to determine dynamic viscosity of Newtonian fluids using standard capillary electrophoresis (CE) equipment. CE is an analytical method routinely used for the separation and characterization of proteins. In our set-up, the capillary is filled with the test sample, and a constant pressure is applied. A small aliquot of riboflavin is subsequently loaded into the capillary and used as a dye to monitor movement of protein samples. Migration time of the riboflavin peak moving through the filled capillary is converted to the viscosity by applying the Hagen-Poiseuille's law. The instrument is operated without using an electrical field. Repeatability, robustness, linearity, and reproducibility were demonstrated for different capillary lots and instruments, as well as for different capillary lengths and diameters. Accuracy was verified by comparing the viscosity data obtained by CE instrumentation with those obtained by plate/cone rheometry. The suitability of the method for protein formulations was demonstrated, and limitations were discussed. Typical viscosities in the range of 5-40mPas were reliably measured with this method. Advantages of the CE instrumentation-based method included short measurement times (1-15min), small sample volumes (few microliters) for a capillary with a diameter of 50μm and a length of 20.5cm as well as potential to be suitable for high-throughput measurements. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Identification of Genetic on Blood Serum Protein of Prolific Ewes

    Science.gov (United States)

    Sutiyono; Ondho, Y. S.; Setiatin, E. T.; Sutopo; Laily, A. N.; Prasetyowati, D. E.; Noviani, F.

    2018-02-01

    The aim of the research was to identify the genetic specification of blood plasma protein in ewes that are prolific. The material of study of local sheep in Bawen and Jambu Sub-district of Semarang Regency is 132 which is determined by purposive sampling that have been give lambing three times. Ewes were divided into three groups that always has a single child (L1), ever had twins (L2) and twins more than two (LM2). Blood sampling was performed using dispossible syringe in jugular vein as much as 5 ml per ewe. Blood plasma was analyzed by Polyacrylamide Gel Electrophoresis-Thin Layer (PAGETLE) method in Biochemistry Laboratory of Veterinary Faculty of Gadjah Mada University. Data analysis is using descriptive statistics and the laws of equilibrium Hardy-Weberg. The research parameters were comparison type of ewes and frequency genetic of protein of blood serum. The results showed that the parent comparisons of L1, L2 and LM2 were 66 (50.00%), 49 (37.12%) and 17 (12.88%), respectively. The frequency genes haven a high propensity to relationship of prolificacy nature parent are Pal2, AlbB, CPF, TFB, PTFS and AmlB on pointes, 67.65, 55.88, 91.17, 70.59, 79.41 and 91.18%. Conclusion the mostly LM2 ewes have genotypes Pal1Pal2, AlbBAlbC, CpFCpF, TfATfB, PtfSPtfS and AmlBAmlB whit frequency are 52.94%, 52.94%, 88.24, 47.06, 64.71 and 88.24% respectively.

  2. Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas

    Science.gov (United States)

    Flint, Mark; Matthews, Beren J.; Limpus, Colin J.; Mills, Paul C.

    2015-01-01

    Biochemical and haematological parameters are increasingly used to diagnose disease in green sea turtles. Specific clinical pathology tools, such as plasma protein electrophoresis analysis, are now being used more frequently to improve our ability to diagnose disease in the live animal. Plasma protein reference intervals were calculated from 55 clinically healthy green sea turtles using pulsed field electrophoresis to determine pre-albumin, albumin, α-, β- and γ-globulin concentrations. The estimated reference intervals were then compared with data profiles from clinically unhealthy turtles admitted to a local wildlife hospital to assess the validity of the derived intervals and identify the clinically useful plasma protein fractions. Eighty-six per cent {19 of 22 [95% confidence interval (CI) 65–97]} of clinically unhealthy turtles had values outside the derived reference intervals, including the following: total protein [six of 22 turtles or 27% (95% CI 11–50%)], pre-albumin [two of five, 40% (95% CI 5–85%)], albumin [13 of 22, 59% (95% CI 36–79%)], total albumin [13 of 22, 59% (95% CI 36–79%)], α- [10 of 22, 45% (95% CI 24–68%)], β- [two of 10, 20% (95% CI 3–56%)], γ- [one of 10, 10% (95% CI 0.3–45%)] and β–γ-globulin [one of 12, 8% (95% CI 0.2–38%)] and total globulin [five of 22, 23% (8–45%)]. Plasma protein electrophoresis shows promise as an accurate adjunct tool to identify a disease state in marine turtles. This study presents the first reference interval for plasma protein electrophoresis in the Indo-Pacific green sea turtle. PMID:27293722

  3. Haematocrit, Haemoglobin, Total Protein And Whole Blood ...

    African Journals Online (AJOL)

    The study was performed on a total of one hundred and nineteen apparently healthy mallards, reared under the traditional extensive management system with the aim of establishing the base-line values of packed cell volume (PCV), haemoglobin (Hb), total protein (TP) and whole blood coagulation time (WBCT) of these ...

  4. Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis.

    Science.gov (United States)

    Antonioli, Paolo; Mendieta, Martha E; Sebastiano, Roberto; Citterio, Attilio; Peltre, Gabriel; Busnel, Jean-Marc; Descroix, Stephanie; Candiano, Giovanni; Righetti, Pier Giorgio

    2006-03-20

    Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.

  5. Proteomic analysis of protein expression profiles during Caenorhabditis elegans development using two-dimensional difference gel electrophoresis.

    Science.gov (United States)

    Tabuse, Yo; Nabetani, Takuji; Tsugita, Akira

    2005-07-01

    Coordinated protein expression is critical for the normal execution of animal development. To obtain overall proteome profiles during animal development, a small free-living soil nematode, Caenorhabditis elegans, was used as a model and the developmental changes of protein expressions were analyzed using two-dimensional difference gel electrophoresis. Protein samples from six developmental stages were prelabeled with fluorescent cyanine dyes and separated on two-dimensional electrophoresis gels. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the developmental expression profiles of 231 spots representing 165 proteins. About a quarter of the identified proteins were expressed in multiple spots with different isoelectric points, suggesting a certain proportion of proteins were variously modified. This notion was supported by the observation that about a third of the multispot proteins were stained positive for a phosphoprotein specific dye. While a fairly large number of the proteins showed little alteration in their expression profiles during development, about 40 proteins were found to be significantly either up- or down-regulated between the embryos and newly hatched L1 larvae. Down-regulated proteins included those related to the cell cycle such as MCM-7, PCN-1, and the mitotic checkpoint protein, while up-regulated proteins included structural proteins such as actins, LEV-11, DIM-1, VAB-21, metabolic enzymes such as ATP synthase, ALH-12, fluctose-1,6-bisphosphate aldolase and GPD-3, and galectins. A standard proteome map was obtained where the defects in the mutations of developmental genes and the effects of reagents on the development in C. elegans were analyzed.

  6. Streamlined sign-out of capillary protein electrophoresis using middleware and an open-source macro application.

    Science.gov (United States)

    Mathur, Gagan; Haugen, Thomas H; Davis, Scott L; Krasowski, Matthew D

    2014-01-01

    Interfacing of clinical laboratory instruments with the laboratory information system (LIS) via "middleware" software is increasingly common. Our clinical laboratory implemented capillary electrophoresis using a Sebia(®) Capillarys-2™ (Norcross, GA, USA) instrument for serum and urine protein electrophoresis. Using Data Innovations Instrument Manager, an interface was established with the LIS (Cerner) that allowed for bi-directional transmission of numeric data. However, the text of the interpretive pathology report was not properly transferred. To reduce manual effort and possibility for error in text data transfer, we developed scripts in AutoHotkey, a free, open-source macro-creation and automation software utility. Scripts were written to create macros that automated mouse and key strokes. The scripts retrieve the specimen accession number, capture user input text, and insert the text interpretation in the correct patient record in the desired format. The scripts accurately and precisely transfer narrative interpretation into the LIS. Combined with bar-code reading by the electrophoresis instrument, the scripts transfer data efficiently to the correct patient record. In addition, the AutoHotKey script automated repetitive key strokes required for manual entry into the LIS, making protein electrophoresis sign-out easier to learn and faster to use by the pathology residents. Scripts allow for either preliminary verification by residents or final sign-out by the attending pathologist. Using the open-source AutoHotKey software, we successfully improved the transfer of text data between capillary electrophoresis software and the LIS. The use of open-source software tools should not be overlooked as tools to improve interfacing of laboratory instruments.

  7. Streamlined sign-out of capillary protein electrophoresis using middleware and an open-source macro application

    Directory of Open Access Journals (Sweden)

    Gagan Mathur

    2014-01-01

    Full Text Available Background: Interfacing of clinical laboratory instruments with the laboratory information system (LIS via "middleware" software is increasingly common. Our clinical laboratory implemented capillary electrophoresis using a Sebia; Capillarys-2™ (Norcross, GA, USA instrument for serum and urine protein electrophoresis. Using Data Innovations Instrument Manager, an interface was established with the LIS (Cerner that allowed for bi-directional transmission of numeric data. However, the text of the interpretive pathology report was not properly transferred. To reduce manual effort and possibility for error in text data transfer, we developed scripts in AutoHotkey, a free, open-source macro-creation and automation software utility. Materials and Methods: Scripts were written to create macros that automated mouse and key strokes. The scripts retrieve the specimen accession number, capture user input text, and insert the text interpretation in the correct patient record in the desired format. Results: The scripts accurately and precisely transfer narrative interpretation into the LIS. Combined with bar-code reading by the electrophoresis instrument, the scripts transfer data efficiently to the correct patient record. In addition, the AutoHotKey script automated repetitive key strokes required for manual entry into the LIS, making protein electrophoresis sign-out easier to learn and faster to use by the pathology residents. Scripts allow for either preliminary verification by residents or final sign-out by the attending pathologist. Conclusions: Using the open-source AutoHotKey software, we successfully improved the transfer of text data between capillary electrophoresis software and the LIS. The use of open-source software tools should not be overlooked as tools to improve interfacing of laboratory instruments.

  8. Structural analysis of protein complexes with sodium alkyl sulfates by small-angle scattering and polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Ospinal-Jiménez, Mónica; Pozzo, Danilo C

    2011-02-01

    Small-angle X-ray (SAXS) and neutron (SANS) scattering is used to probe the structure of protein-surfactant complexes in solution and to correlate this information with their performance in gel electrophoresis. Proteins with sizes between 6.5 to 116 kDa are denatured with sodium alkyl sulfates (SC(x)S) of variable tail lengths. Several combinations of proteins and surfactants are analyzed to measure micelle radii, the distance between micelles, the extension of the complex, the radius of gyration, and the electrophoretic mobility. The structural characterization shows that most protein-surfactant complexes can be accurately described as pearl-necklace structures with spherical micelles. However, protein complexes with short surfactants (SC(8)S) bind with micelles that deviate significantly from spherical shape. Sodium decyl (SC(10)S) and dodecyl (SC(12)S, more commonly abbreviated as SDS) sulfates result in the best protein separations in standard gel electrophoresis. Particularly, SC(10)S shows higher resolutions for complexes of low molecular weight. The systematic characterization of alkyl sulfate surfactants demonstrates that changes in the chain architecture can significantly affect electrophoretic migration so that protein-surfactant structures could be optimized for high resolution protein separations.

  9. Differences between fertilized and unfertilized chicken egg white proteins revealed by 2-dimensional gel electrophoresis-based proteomic analysis.

    Science.gov (United States)

    Qiu, Ning; Liu, Wen; Ma, Meihu; Zhao, Lei; Li, Yuqi

    2013-03-01

    The egg white protein alterations during the early phase of chicken embryonic development were recently reported by our laboratory. Nevertheless, the original albumen differences between fresh unfertilized and fertilized chicken eggs have not been investigated. By using 2-dimensional gel electrophoresis (2-DE), coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) method, 1 ovalbumin protein spot as well as 6 ovalbumin-related protein Y spots were identified showing more than 10-fold differences (P egg whites. Six of these protein spots represented higher intensity in fertilized eggs through 2-DE analysis. It was thus concluded that ovalbumin protein family, especially ovalbumin-related protein Y, may play an important role in embryonic development, which still needs to be validated. This finding will provide insight into embryogenesis to improve our understanding of the functions of ovalbumin family proteins in regulating or supporting embryonic development.

  10. BLOOD PROTEIN TRANSFERRIN POLYMOROPHISM IN BLACK BENGAL GOAT

    Directory of Open Access Journals (Sweden)

    Rajesh Paul

    2017-12-01

    Full Text Available The present investigation was carried out with an aim to explore the polymorphism of a blood protein tranferrin using starch gel electrophoresis technique in a total of unrelated 199 Black Bengal goats available in four different districts of West Bengal, India. Banding patterns of transferrin in starch gel revealed six phenovariants TfAA, TfAB, TfBC, TfBB, TfAC and TfCC comprising of three allelomorphs, TfA, TfB and TfC. The genotype frequencies were found to be observed 0.211, 0.347, 0.136, 0.106, 0.136 and 0.065 for six genotypes and the allelic frequencies were 0.452, 0.347 and 0.201 for three alleles, respectively. Result of Chi-square test revealed that the population under study was in Hardy Weinberg Equilibrium. There were polymorphism in Transferrin protein and the presence of differences among the frequencies of the three alleles by categories could be a source of genetic variation in Black Bengal goat.

  11. Protein Electrophoresis/Immunofixation Electrophoresis

    Science.gov (United States)

    ... and Iron-binding Capacity (TIBC, UIBC) Trichomonas Testing Triglycerides Troponin Tryptase Tumor Markers Uric Acid Urinalysis Urine ... Ceruloplasmin Copper-containing enzyme, involved in normal iron metabolism Beta-globulin Transferrin Iron transport and delivery to ...

  12. Speciation of iodine-containing proteins in Nori seaweed by gel electrophoresis laser ablation ICP-MS.

    Science.gov (United States)

    Romarís-Hortas, V; Bianga, J; Moreda-Piñeiro, A; Bermejo-Barrera, P; Szpunar, J

    2014-09-01

    An analytical approach providing an insight into speciation of iodine in water insoluble fraction of edible seaweed (Nori) was developed. The seaweed, harvested in the Galician coast (Northwestern Spain), contained 67.7±1.3 μg g(-1) iodine of which 25% was water soluble and could be identifies as iodide. Extraction conditions of water insoluble residue using urea, NaOH, SDS and Triton X-100 were investigated. The protein pellets obtained in optimized conditions (after precipitation of urea extracts with acetone), were digested with trypsin and protease XIV. Size exclusion chromatography-ICP-MS of both enzymatic digests demonstrated the occurrence of iodoaminoacids putatively present in proteins. Intact proteins could be separated by gel electrophoresis after an additional extraction of the protein extract with phenol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with laser ablation ICP-MS detection of (127)I indicated the presence of iodine in protein bands corresponding to molecular masses of 110 kDa, 40 kDa, 27 kDa, 20 kDa and 10 kDa. 2D IEF-SDS PAGE with laser ablation ICP-MS (127)I imaging allowed the detection of 5 iodine containing protein spots in the alkaline pI range. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. [Choice of the optimal method of numerical analysis in taxonometric study of mollicutes based on electrophoresis of their proteins].

    Science.gov (United States)

    Skripal', I G; Karpov, A V; Malinovskaia, L P; Sadovnikov, Iu S

    1989-01-01

    Different procedures of numerical analysis are evaluated to select the most convenient of them for processing the results of mycoplasm protein electrophoresis in polyacrylamide gel (EPAAG) when solving problems of their taxonomy and systematics. This aim is satisfied most of all by the method of Sokal which permits revealing relations at the rank of species, genus and higher taxons. This method as well as the approaches developed by the authors for transforming results of EPAAG of proteins into the numerical form are recommended to improve the systematics of mollicutes with application of computers.

  14. A new multiphasic buffer system for benzyldimethyl-n-hexadecylammonium chloride polyacrylamide gel electrophoresis of proteins providing efficient stacking.

    Science.gov (United States)

    Kramer, Michael L

    2006-02-01

    Acidic PAGE systems using cationic detergents such as benzyldimethyl-n-hexadecylammonium chloride (16-BAC) or CTAB have proven useful for the detection of methoxy esters sensitive to alkaline pH, resolving basic proteins such as histones and membrane proteins. However, the interesting phosphate-based system suffered from poor stacking, resulting in broadened bands and long running times. Therefore, a new 16-BAC PAGE system based on the theory of moving boundary electrophoresis with properties comparable to the classical SDS-PAGE system was designed. As a result a new multiphasic analytical 16-BAC PAGE system providing efficient stacking and significantly shorter running times is presented here. It is based on acetic acid and methoxyacetic acid as common ion constituents. This PAGE system takes advantage of the additional counter stacking effect due to a cross boundary electrophoresis system resulting from the selected buffer constituents. Furthermore, the concentration of 16-BAC was optimized by determining its previously unknown CMC. Due to efficient focusing of the introduced tracking dye, methyl green, termination of electrophoresis can now be more easily followed as compared to the Schlieren line.

  15. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.

    Energy Technology Data Exchange (ETDEWEB)

    Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P. A.; Vogt, S.; Univ. of Sydney; Northwestern Univ.

    2010-01-01

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

  16. Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Lu, Y; Qi, Y X; Zhang, H; Zhang, H Q; Pu, J J; Xie, Y X

    2013-12-19

    To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

  17. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    Science.gov (United States)

    Sheen, Hyukho

    2016-04-01

    Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization.

    Science.gov (United States)

    Dong, Ming; Choi, Megan; Biggin, Mark D; Jin, Jian

    2012-01-01

    We developed a multichannel gel electrophoresis system that continuously collects fractions as protein bands migrate off the bottom of gel columns. The device uses several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique allows continuous and simultaneous fraction collection from multiple channels at low cost. Using the system with SDS-PAGE, 300 μg samples of protein can be separated and eluted into 48-96 fractions over a mass range of 10-150 kDa in 2.5 h. Each eluted protein can be recovered at 50% efficiency or higher in ∼500 μL. The system can also be used for native gel electrophoresis, but protein aggregation limits the loading capacity to about 50 μg per channel and reduces resolution. This system has the potential to be coupled with mass spectrometry to achieve high-throughput protein identification.

  19. Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

    Science.gov (United States)

    Picariello, Gianluca; De Martino, Alessandra; Mamone, Gianfranco; Ferranti, Pasquale; Addeo, Francesco; Faccia, Michele; Spagnamusso, Salvatore; Di Luccia, Aldo

    2006-03-20

    In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate

  20. Carbon nanotubes-assisted polyacrylamide gel electrophoresis for enhanced separation of human serum proteins and application in liverish diagnosis.

    Science.gov (United States)

    Jiang, Fubin; Wang, Yanan; Hu, Xinfang; Shao, Na; Na, Na; Delanghe, Joris R; Ouyang, Jin

    2010-11-01

    The application of pore-gradient polyacrylamide gel electrophoresis (PG-PAGE) incorporated with carbon nanotube modified by Triton X-100 and carboxylation so as to improve the separation of human serum proteins is reported. The novel PG-PAGE was made by adding water-soluble single-walled carbon nanotubes (CNTs) when preparing the polyacrylamide gel. Significant improvements in separation of complement C3 protein and haptoglobin (Hp) in human serum were achieved. It was estimated that the interactions between the hydrophilic groups on the proteins and the surface of the CNTs result in different adsorption kinetics of complement C3 and Hp subtype on the nanoparticles incorporated in the gel, thus enhancing the separation of the two proteins in serum. This new CNT matrix-assisted PG-PAGE method for enhanced separation of complement C3 and Hp in human serum was successfully applied to distinguish the samples from liverish patients and healthy people.

  1. Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Sano, Osamu; Kunikata, Toshio; Kohno, Keizo; Iwaki, Kanso; Ikeda, Masao; Kurimoto, Masashi

    2004-01-14

    In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.

  2. Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Park, Chul; Helm, Richard F; Novak, John T

    2008-12-01

    The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study.

  3. Evaluation of different protein extraction methods for banana (Musa spp.) root proteome analysis by two-dimensional electrophoresis.

    Science.gov (United States)

    Vaganan, M Mayil; Sarumathi, S; Nandakumar, A; Ravi, I; Mustaffa, M M

    2015-02-01

    Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.

  4. Heterogeneity of a labeled tumor surface protein from a murine lung carcinoma demonstrated by two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Eisinger, R.W. (Univ. of Tennessee, Oak Ridge); Kennel, S.J.

    1981-03-01

    Heterogeneity of a tumor surface protein (designated TSP-180) has been demonstrated by two-dimensional electrophoresis. Line 1 carcinoma cells derived from a spontaneous alveolar carcinoma of BALB/c mice were labeled externally with /sup 125/I by use of lactoperoxidase or metabolically with (/sup 3/H)-leucine before cell proteins were solubilized with Triton X-100 detergent. Immunoprecipitates prepared with heterologous antisera allowed comparison of two-dimensional patterns of line 1 surface proteins labeled with /sup 125/I or /sup 3/H. The isoelectric point of /sup 125/I-labeled TSP-180 was heterogeneous and varied between 6.1 and 6.3. Treatment with neuraminidase shifted the pI values to between 5.9 and 6.1 and reduced, but did not eliminate, the banding heterogeneity. These data show that charge heterogeneity due to sialization, as well as other factors, exists in TSP-180.

  5. Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Olena Zakharchenko

    2011-01-01

    Full Text Available Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE. This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.

  6. Application of zwitterionic detergent to the solubilization of Klebsiella pneumoniae outer membrane proteins for two-dimensional gel electrophoresis.

    Science.gov (United States)

    Bednarz-Misa, I; Serek, P; Dudek, B; Pawlak, A; Bugla-Płoskońska, G; Gamian, A

    2014-12-01

    Klebsiella pneumoniae is a frequent cause of nosocomial respiratory, urinary and gastrointestinal tract infections and septicemia with the multidrug-resistant K. pneumoniae being a major public health concern. Outer membrane proteins (OMPs) are important virulence factors responsible for the appropriate adaptation to the host environment. They constitute of the antigens being the first in contact with infected organism. However, K. pneumoniae strains are heavily capsulated and it is important to establish the OMPs isolation procedure prior to proteomics extensive studies. In this study we used Zwittergent Z 3-14® as a detergent to isolate the OMPs from K. pneumoniae cells and resolve them using two-dimensional electrophoresis (2-DE). As a result we identified 134 protein spots. The OMPs identified in this study are possible candidates for the development of a protein-based vaccine against K. pneumoniae infections. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. On-Line Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry for Preconcentration and Clean-Up of Peptides and Proteins.

    Science.gov (United States)

    Benavente, Fernando; Medina-Casanellas, Silvia; Giménez, Estela; Sanz-Nebot, Victoria

    2016-01-01

    One of the major drawbacks of capillary electrophoresis (CE) and other microscale separation techniques, for the analysis of low abundant peptides and proteins in complex samples, are the poor concentration limits of detection. Several strategies have been developed to improve CE sensitivity. Here, we describe an on-line solid-phase extraction capillary electrophoresis mass spectrometry method with a commercial C18 sorbent for clean-up and preconcentration of neuropeptides from highly diluted biological samples.

  8. Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis.

    Science.gov (United States)

    An, Philip; Winters, Dwight; Walker, Kenneth W

    2016-04-01

    Modern molecular genetics technology has made it possible to swiftly sequence, clone and mass-produce recombinant DNA for the purpose of expressing heterologous genes of interest; however, recombinant protein production systems have struggled to keep pace. Mammalian expression systems are typically favored for their ability to produce and secrete proteins in their native state, but bacterial systems benefit from rapid cell line development and robust growth. The primary drawback to prokaryotic expression systems are that recombinant proteins are generally not secreted at high levels or correctly folded, and are often insoluble, necessitating post-expression protein folding to obtain the active product. In order to harness the advantages of prokaryotic expression, high-throughput methods for executing protein folding screens and the subsequent analytics to identify lead conditions are required. Both of these tasks can be accomplished using a Biomek 3000 liquid handling robot to prepare the folding screen and to subsequently prepare the reactions for assessment using Caliper microfluidic capillary electrophoresis. By augmenting a protein folding screen with automation, the primary disadvantage of Escherichia coli expression has been mitigated, namely the labor intensive identification of the required protein folding conditions. Furthermore, a rigorous, quantitative method for identifying optimal protein folding buffer aids in the rapid development of an optimal production process. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Differences in serum protein 2D gel electrophoresis patterns of Przewalski's (Mongolian wild horse) and thoroughbred horses.

    Science.gov (United States)

    Barsuren, Enkhbolor; Namkhai, Bandi; Kong, Hong Sik

    2015-04-01

    The objective of this study was to assess differences in serum protein expression profiles of Przewalski's (Mongolian wild horse) and thoroughbred horses using proteome analysis. The serum proteins were separated by two-dimensional electrophoresis (2-DE) and five different gene products were identified. Proteins represented by the five spots were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS technology. The identities of all proteins were deduced based on their similarity to proteins in the human plasma protein database. Three proteins (a haptoglobin-2 alpha glycoprotein and two haptoglobin-2beta glycoproteins with different accession numbers) were downregulated in Przewalski's horse sera compared to thoroughbred horse sera. Moreover, two proteins (tetraspanin-18 and pM5) were upregulated in Przewalski's horses compared to thoroughbred horses. Haptoglobin-2 alpha and haptoglobin-2beta may serve as candidate molecules in future studies of inflammation, coagulation, immune modulation and pro-oxidant and antioxidant activity with consequential effects on the entire metabolism of the horse. © 2014 Japanese Society of Animal Science.

  10. Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye.

    Science.gov (United States)

    Fujii, Kazuyasu; Kondo, Tadashi; Yokoo, Hideki; Okano, Tetsuya; Yamada, Masayo; Yamada, Tesshi; Iwatsuki, Keiji; Hirohashi, Setsuo

    2006-03-01

    CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.

  11. A comparative analysis of phloem exudate proteins from Cucumis melo, Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing.

    Science.gov (United States)

    Sabnis, D D; Hart, J W

    1976-01-01

    Proteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.

  12. Genetic variability in the sable (Martes zibellina L.) with respect to genes encoding blood proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kashtanov, S.N. [Vavilov Institute of General Genetics, Moscow (Russian Federation); Kazakova, T.I. [Afanas`ev Scientific Research Institute for Breeding of Fur-Bearing Animals, Moscow (Russian Federation)

    1995-02-01

    Electrophoresis of blood proteins was used to determine, for the first time, the level of genetic variability of certain loci in the sable (Martes zibellina L., Mustelidae). Variation of 23 blood proteins encoded by 25 genes was analyzed. Polymorphism was revealed in six genes. The level of heterozygosity was estimated at 0.069; the proportion of polymorphic loci was 24%. Data on the history of the sable population maintained at the farm, on geographical distribution of natural sable populations, and on the number of animals selected for reproduction in captivity is presented. The great number of animals studies and the extensive range of natural sable populations, on the basis of which the population maintained in captivity was obtained, suggest that the results of this work can be used for estimating the variability of the gene pool of sable as a species. 9 refs., 2 figs., 1 tab.

  13. Application of Microchip Electrophoresis for Clinical Tests

    Science.gov (United States)

    Yatsushiro, Shouki; Kataoka, Masatoshi

    Microchip electrophoresis has recently attracted much attention in the field of nuclear acid analysis due to its high efficiency, ease of operation, low consumption of samples and reagents, and relatively low costs. In addition, the analysis has expanded to an analytical field like not only the analysis of DNA but also the analysis of RNA, the protein, the sugar chain, and the cellular function, etc. In this report, we showed that high-performance monitoring systems for human blood glucose levels and α-amylase activity in human plasma using microchip electrophoresis.

  14. Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis

    International Nuclear Information System (INIS)

    Carrasco, L.; Bravo, R.

    1986-01-01

    The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with [ 3 H]glucosamine were detected in vaccinia-infected HeLa cells

  15. Characterisation of ribosomal proteins from HeLa and Krebs II mouse ascites tumor cells by different two-dimensional polyacrylamide gel electrophoresis techniques

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H

    1978-01-01

    electrophoresis; 2. two-dimensional gel electrophoresis at pH 4.K/pH 8.6 in SDS. The molecular weights for 40S proteins ranged from 10,000 to 39,000 dalton (number average molecular weight: 21,000). The molecular weights for the 60S proteins ranged from 14,000 to 44,000 dalton (number average molecular weight: 23...... using the pH 4.5/pH 8.6 SDS system. The molecular weights Krebs II ascites and HeLa ribosomal proteins are compared with those obtained by other authors for different mammalian species....

  16. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system....... A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots...

  17. Effects of Storage and Granary Weevil Infestation on Gel Electrophoresis and Protein Solubility Properties of Hard and Soft Wheat Flours.

    Science.gov (United States)

    Keskin, Sule; Yalçin, Erkan; Özkaya, Hazim

    2018-02-24

    The objective of this study was to investigate the effects of storage and granary weevil, Sitophilus granarius (L.; Coleoptera: Curculionidae), infestation on pH, protein solubility (PS) and gel electrophoresis properties of meal and roller-milled flours of hard (Ceyhan-99 cv.) and soft (Eser cv.) wheat cultivars, respectively, after 6 mo of storage. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique was applied for studying the electrophoretic properties. Hard and soft wheats were infested with non-sexed S. granarius at a rate of two adults/ kg, and stored for 6 mo at 30 ± 1°C and 70 ± 5% RH. The pest-free wheat samples were used as control. The infested and its control samples were collected monthly, and after cleaning the granary weevils, they were hammer-milled or roller-milled in order to get meal flours and roller-milled flours, respectively. The effect of infestation on the storage proteins was more obvious in meal flours than that of the roller-milled flours. Granary weevil feeding resulted secreting of hydrolyzing enzymes and increased the acidity of flours; subsequently the breaking and releasing of some storage proteins generally caused a decrease in pH and an increase in PS values of the meal flours of wheat cultivars. SDS-PAGE results generally indicated that towards the end of storage, the insect population, that greatly increased, caused minor protein depletions resulting decreasing protein band intensities between 113 and 58 kDa of hard wheat meal flour and 101 and 40 kDa of soft wheat roller-milled flour. Consequently, the potential effect of changes probably occurred in high molecular weight glutenin subunits of both wheat cultivars.

  18. Metal-binding proteins scanning and determination by combining gel electrophoresis, synchrotron radiation X-ray fluorescence and atomic spectrometry.

    Science.gov (United States)

    Verbi, F M; Arruda, S C C; Rodriguez, A P M; Pérez, C A; Arruda, M A Z

    2005-02-28

    In the present work, protein bands from in vitro embriogenic callus (Citrus sinensis L. Osbeck) were investigated using micro-synchrotron radiation X-ray fluorescence (muSR-XRF) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Metal-binding protein quantification was done after microwave oven decomposition of gel by synchrotron radiation total reflection X-ray fluorescence (SR-TXRF), flame atomic absorption spectrometry (FAAS) and flame atomic emission spectrometry (FAES). According to the analysis of the protein bands, it is possible to observe that both 81 and ca. 14 kDa proteins present different Fe signal intensity at different positions. The analysis of 53 kDa protein, showed even more interesting results. Besides Fe, the muSR-XRF experiments indicate the presence of Ca, Cu, K and Zn. Chemical elements such as Cu, K, Fe and Zn were determined by SR-TXRF, Mg by FAAS and Na by FAES. Ca was determined by SR-TXRF and FAAS only for accuracy check. In the mineralised protein bands of 81 and around 14 kDa band, only Fe was determined (105 and 21.8 microg g(-1)). For those protein bands (86-ca. 14 kDa) were determined, Ca, K, Cu and Zn in a wide concentration range (42.4-283, 2.47-96.8, 0.91-15.9 and 3.39-29.7 microg g(-1), respectively).

  19. Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Henningsen, Robert; Gale, Brenda L; Straub, Kenneth M; DeNagel, Diane C

    2002-11-01

    Comparative analysis has long been utilized in biological research to interpret protein interactions in both drug naïve versus drug challenged and normal versus diseased tissues. The technology of proteomics today allows researchers to provide insight into old and still open questions related to biological mechanisms while offering the opportunity to discover novel details in cellular lifecycles. Perhaps the most powerful way to execute these differential displays is in the combination of two-dimensional (2-D) gel electrophoresis and mass spectrometry. While these two techniques together are well suited for abundant and soluble proteins found in cells, rare proteins and integral membrane proteins are still problematic. Recently, a series of novel zwitterionic detergents has been reported in the literature that shows a substantial improvement in solubilizing integral membrane proteins. We show that the amidosulfobetaine, 4-octylbenzol amidosulfobetaine, is better than 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) at solubilizing both an ion channel and a G-protein coupled receptor (GPCR), while another amidosulfobetaine, myristic amidosulfobetaine (ASB-14), was better than CHAPS at solubilizing a GPCR. Neither membrane protein was visible after staining with colloidal Coomassie blue, silver nor Sypro Ruby. However, a comparison against a duplicate immunoblot allowed for the localization and identification of the ion channel from a 2-D gel by liquid chromatography-tandem mass spectrometry.

  20. A comparative method for protein extraction and 2D-gel electrophoresis from different tissues of Cajanus cajan

    Directory of Open Access Journals (Sweden)

    Nisha eSingh

    2015-08-01

    Full Text Available Pigeonpea is an important legume crop with high protein content. However, it is often subjected to various abiotic and biotic stresses. Proteomics is a state-of-the-art technique used to analyze the protein profiling of a tissue for deciphering the molecular entities that could be manipulated for developing crops resistant to these stresses. In this context, developing a comprehensive proteome profile from different vegetative and reproductive tissues has become mandatory. Although several protein extraction protocols from different tissues of diverse plant species have been reported, there is no report for pigeonpea. Here, we report tissue-specific protein extraction protocols representing vegetative (young leaves, and reproductive (flowers and seeds organs and their subsequent analysis on 2-dimensional gel electrophoresis. The study explicitly demonstrated that the efficacy of a particular protein extraction protocol is dependent on the different tissues, such as leaves, flowers and seeds that differ in their structure and metabolic constituents. For instance, phenol-based protocol showed an efficacy towards higher protein yield, better spot resolution and a minimal streaking on 2-DE gel for both leaves and flowers. Protein extraction from seeds was best achieved by employing phosphate-TCA-acetone protocol.

  1. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

    Directory of Open Access Journals (Sweden)

    Duban-Deweer Sophie

    2010-11-01

    Full Text Available Abstract Background Brain capillary endothelial cells (BCECs form the physiological basis of the blood-brain barrier (BBB. The barrier function is (at least in part due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein. Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

  2. Improving sodium dodecyl sulfate polyacrylamide gel electrophoresis detection of low-abundance protein samples by rapid freeze centrifugation.

    Science.gov (United States)

    Virgen-Ortíz, J J; Ibarra-Junquera, V; Escalante-Minakata, P; Osuna-Castro, J A; Ornelas-Paz, J de J; Mancilla-Margalli, N A; Castañeda-Aguilar, R L

    2013-12-15

    This work presents a rapid and simple freeze centrifugation method to concentrate dilute protein solutions for detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Coomassie blue staining. Moreover, a simple way to assemble a cryoconcentration device is presented, and its use is discussed. Commercial purified protein standard and an enzyme with high fructosyltransferase (FTase) activity, coming from target fractions obtained by chromatographic separation, were used as an example. FTase, coming directly from the chromatographic fractions, was difficult to view through SDS-PAGE analysis; however, it was easily visualized, and its activity was enhanced, after the application of the freeze centrifugation protocol presented here. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

    Science.gov (United States)

    Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

    2014-07-04

    Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

  4. [Study on elimination of 3-chloro-1,2-propanediol in hydrolyzed vegetable protein by capillary electrophoresis with electrochemical detection].

    Science.gov (United States)

    Xing, Xiaoping; Cao, Yuhua

    2006-03-01

    A method was developed to estimate 3-chloro-1,2-propanediol (3-MCPD) in hydrolyzed vegetable protein (HVP) by capillary electrophoresis with electrochemical detection. The optimum conditions were investigated to determine 3-MCPD in hydrolyzed vegetable protein. In 30 mmol/L borax (pH 9.24) running buffer, separation of 3-MCPD from glycerol was achieved. A 328 microm copper-disk electrode used as the working electrode exhibited a good response at + 0.65 V (vs. saturated calomel electrode (SCE)) in 0.05 mol/L sodium hydroxide solution. The effects of pH value, temperature and hydrolysis time on the elimination of 3-MCPD in hydrolyzed vegetable protein were studied. By adjusting hydrolyzed vegetable protein to pH value of 8.0 and heating up for 1 h at 90 degrees C, the content of 3-MCPD in hydrolyzed vegetable protein could be controlled under the level of 1.0 mg/kg, which coincides with the criterion of food safety in China.

  5. Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry.

    Science.gov (United States)

    Santos, Simone G; Diniz, Cláudio G; Silva, Vânia L; Lima, Francisca L; Andrade, Hélida M; Chapeaurouge, Donat A; Perales, Jonas; Serufo, José Carlos; Carvalho, Maria Auxiliadora R; Farias, Luiz M

    2012-02-01

    Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.

    Science.gov (United States)

    Fernández-García, Aurora; Alvarez-García, Gema; Marugán-Hernández, Virginia; García-Lunar, Paula; Aguado-Martínez, Adriana; Risco-Castillo, Verónica; Ortega-Mora, Luis M

    2013-07-01

    Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, Presult, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

  7. Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana

    Directory of Open Access Journals (Sweden)

    HADIWIYONO

    2011-01-01

    Full Text Available Hadiwiyono, Widada J, Subandiyah S, Fegan F (2011 Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana. Biodiversitas 12: 12-16. Blood disease bacterium (BDB is the most important pathogen of bananas in Indonesia. In some field, the disease incidence reaches over 80%. Epidemiologically, the disease is similar to moko disease in South America and bugtok disease in the Philippines caused by Ralstonia solanacearum race 2. However, BDB is different in phenotype and genotype from the two diseases. Previously BDB was limited in South Sulawesi since 1920s – 1980s and recently was reported in 27 of 30 provinces in Indonesia. Pulsed-Field Gel Electrophoresis (PFGE is a genomic DNA fingerprinting method, which employs rare cutting restriction endonucleases to digest genome prior to electrophoresis using specialized condition to separate of large DNA fragments. The results showed that PFGE analysis was a discriminative tool to study the genetic diversity of BDB. Based on the PFGE analysis, BDB isolates obtained from different localities in Yogyakarta and Central Java were quit diverse.

  8. Interconverting conformations of variants of the human amyloidogenic protein beta2-microglobulin quantitatively characterized by dynamic capillary electrophoresis and computer simulation

    DEFF Research Database (Denmark)

    Heegaard, Niels H H; Jørgensen, Thomas J D; Cheng, Lei

    2006-01-01

    Capillary electrophoretic separation profiles of cleaved variants of beta2-microglobulin (beta2m) reflect the conformational equilibria existing in solutions of these proteins. The characterization of these equilibria is of interest since beta2m is responsible for amyloid formation in dialysis...... a unified theory for dynamic chromatography and dynamic electrophoresis. The results are correlated with the outcome of independent experiments based on mass spectrometric measurement of H/D exchange. This study illustrates that dynamic capillary electrophoresis is suitable for the investigation...... of the interconversion of protein conformations of amyloidogenic molecules and is not only restricted to ideal model compounds....

  9. Molecular phylogeny of the hominoid primates as indicated by two-dimensional protein electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Goldman, D.; Giri, P.R.; O' Brien, J.O.

    1987-05-01

    A molecular phylogeny for the hominoid primates was constructed by using genetic distances from a survey of 383 radiolabeled fibroblast polypeptides resolved by two-dimensional electrophoresis (2DE). An internally consistent matrix of Nei genetic distances was generated on the basis of variants in electrophoretic position. The derived phylogenetic tree indicated a branching sequence, from oldest to most recent, of cercopithecoids (Macaca fascicularis), gibbon-siamang, orangutan, gorilla, and human-chimpanzee. A cladistic analysis of 240 electrophoretic characters that varied between ape species produced an identical tree. Genetic distance measures obtained by 2DE are largely consistent with those generated by other molecular procedures. In addition, the 2DE data set appears to resolve the human-chimpanzee-gorilla trichotomy in favor of a more recent association of chimpanzees and humans.

  10. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    Science.gov (United States)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  11. Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis.

    Science.gov (United States)

    Rodrigues, Silas Pessini; Ventura, José Aires; Zingali, R B; Fernandes, P M B

    2009-01-01

    A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE. Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.

  12. A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

    DEFF Research Database (Denmark)

    Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes

    Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

  13. A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

    DEFF Research Database (Denmark)

    Larsen, Martin; Skottrup, Peter; Enghild, Jan J.

    2005-01-01

    Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

  14. Two-dimensional electrophoresis of proteins discriminates aphid clones of Sitobion avenae differing in BYDV-PAV transmission.

    Science.gov (United States)

    Papura, D; Jacquot, E; Dedryver, C A; Luche, S; Riault, G; Bossis, M; Rabilloud, T

    2002-10-01

    Aset of 39 F1 Sitobion avenae clones was obtained by selfing a poorly efficient BYDV-PAV vector clone. These clones were genetically typed by 11 microsatellite loci, and tested for BYDV-PAV4 transmission to barley. The 39 clones displayed a continuum in transmission percentages, from 0% to 88% with a significant clone effect. From this set, two highly efficient (HEV) and two poorly efficient (PEV) vectoring clones were more precisely characterized for transmission of two other PAV isolates. The molecular bases of the lower transmissibility of BYDV-PAV4 by PEV clones and of the aphid vectoring properties were investigated respectively by comparing the sequences corresponding to structural proteins (CP and RTD) of BYDV, and by using proteomic analysis of aphids in two dimensional electrophoresis (2-DE) with immobilized pH gradients (IPG) after an improved protein extraction. Four residues specific to BYDV-PAV4 located in the CP sequence (A(24) and L(130)) or in the RTD region (M(334) and S(456)) could be responsible for the lower transmissibility of this isolate by PEV clones. Among a total of 2150 well-resoluted spots scored on S. avenae proteinic pattern, only twelve proteins were qualitatively or quantitatively different between clones. Four out of them discriminated HEV and PEV groups.

  15. Characterization of several milk proteins in Domestic Balkan donkey breed during lactation, using lab-on-a-chip capillary electrophoresis

    Directory of Open Access Journals (Sweden)

    Gubić Jasmina

    2016-01-01

    Full Text Available Domestic Balkan donkey (Equus asinus asinus is a native donkey breed, primarily found in the northern and eastern regions of Serbia. The objective of the study was to analyze proteins of Domestic Balkan donkey milk during the lactation period (from the 45th to the 280th day by applying Lab-on-a-Chip electrophoresis. The chip-based separations were performed on the Agilent 2100 Bioanalyzer in combination with the Protein 80 Plus Lab Chip kit. The protein content of Domestic Balkan donkey milk during the lactation period of 280 days ranged from 1.40 % to 1.92 % and the content of αs1-casein, αs2-casein, b-casein, α-, β- lactoglobulin, lysozyme, lactoferrin and serum albumin was relatively quantified. Lysozyme (1040-2970 mg/L, α-lactalbumin 12 kDa (1990-2730 mg/L and α-lactalbumin 17.7 kDa (2240-3090 mg/L were found to be the proteins with the highest relative concentrations. [Projekat Ministarstva nauke Republike Srbije, br. III46012

  16. Avoiding acidic region streaking in two-dimensional gel electrophoresis: case study with two bacterial whole cell protein extracts.

    Science.gov (United States)

    Roy, Arnab; Varshney, Umesh; Pal, Debnath

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  17. GENETIC VARIATION IN THREE BREEDS OF INDONESIAN LOCAL DUCKS BASED ON BLOOD AND EGG WHITE PROTEIN POLYMORPHISM

    Directory of Open Access Journals (Sweden)

    S. Johari

    2013-03-01

    Full Text Available A study of genetic variation of four blood protein loci and three egg white loci in three breeds of Indonesian local ducks (Magelang duck = MAD, Tegal duck = TED and Mojosari duck= MOD was investigated by using polyacrilamide gel electrophoresis (PAGE. The result showed that four blood protein loci (Pre-albumin, Albumin, Transferrin and Post-transferrin and three egg white loci (Ovalbumin, Ovoglobulin and Conalbumin were found to be polymorphic in three breeds of local ducks. There was no difference on blood and egg white protein polyimorphism. The average value of heterozygosity on three breeds of local ducks had a genetic potential to determine the controlled mating system and selection to obtain pure line. The closest phylogenetic relationship among the three breeds of local ducks occurred between MAD and TED.

  18. Evaluation of a capillary zone electrophoresis system versus a conventional agarose gel system for routine serum protein separation and monoclonal component typing.

    Science.gov (United States)

    Roudiere, L; Boularan, A M; Bonardet, A; Vallat, C; Cristol, J P; Dupuy, A M

    2006-01-01

    Capillary zone electrophoresis of serum proteins is increasingly gaining impact in clinical laboratories. During 2003, we compared the fully automated capillary electrophoresis (CE) system from Beckman (Paragon CZE 2000) with the method agarose gel electrophoresis Sebia (Hydrasis-Hyris, AGE). This new study focused on the evaluation of analytical performance and a comparison including 115 fresh routine samples (group A) and a series of 97 frozen pathologic sera with suspicion of monoclonal protein (group B). Coefficients of variation (CVs %) for the five classical protein fractions have been reported to be consistenly serum samples (group B), there were 90 in which we detected a monoclonal protein by immunofixation (IF) (immunosubtraction (IS) was not used). AGE and Paragon 2000 failed to detect 7 and 12 monoclonal proteins, respectively, leading to a concordance to 92% for AGE and 87% for Paragon 2000 for identifying electrophoretic abnormalities in this group. Beta-globulin abnormalities and M paraprotein were well detected with Paragon 2000. Only 81% (21 vs 26) of the gammopathies were immunotyped with IS by two readers blinded to the IF immunotype. The Paragon 2000 is a reliable alternative to conventional agarose gel electrophoresis combining the advantages of full automation (rapidity, ease of use and cost) with high analytical performance. Qualified interpretation of results requires an adaptation period which could further improve concordance between the methods. Recently, this CE system has been improved by the manufacturer (Beckman) concerning the migration buffer and detection of beta-globulin abnormalities.

  19. Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius

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    Zheng Xiaojuan

    2009-10-01

    Full Text Available Abstract Background Two-dimensional gel electrophoresis (2-DE is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF, a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS. Results An extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v 3-[(3-cholamidopropyl-dimethylammonio]-1-propanesulfonate (CHAPS, 50 mM dithiothreitol (DTT, 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF, 20 U/ml Deoxyribonuclease I (DNase I, and 0.25 mg/ml Ribonuclease A (RNase A, combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF. When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found. Conclusion These results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

  20. Comparison of protein patterns after two-dimensional gel electrophoresis from leaves of in vitro cultures and seedlings of Rubus chamaemorus L.

    Directory of Open Access Journals (Sweden)

    Barbara Thiem

    2014-01-01

    Full Text Available Proteins from leaves of Rubus chamaemorus propagated in vitro were subjected to miniaturized 2-D electrophoresis. The 2-DE patterns of proteins showed qualitative differences between plants propagated in vitro and control seedlings. More proteins of a high molecular weight were observed in leaves of plants from in vitro culture. A two-dimensional map of proteins from leaves provides detailed data concerning both polymorphism and protein patterns of this species. This makes it possible to start constructing a protein map of R. chamaemorus. The reasons for qualitative differences are discussed.

  1. A visual detection of protein content based on titration of moving reaction boundary electrophoresis.

    Science.gov (United States)

    Wang, Hou-Yu; Guo, Cheng-Ye; Guo, Chen-Gang; Fan, Liu-Yin; Zhang, Lei; Cao, Cheng-Xi

    2013-04-24

    A visual electrophoretic titration method was firstly developed from the concept of moving reaction boundary (MRB) for protein content analysis. In the developed method, when the voltage was applied, the hydroxide ions in the cathodic vessel moved towards the anode, and neutralized the carboxyl groups of protein immobilized via highly cross-linked polyacrylamide gel (PAG), generating a MRB between the alkali and the immobilized protein. The boundary moving velocity (V(MRB)) was as a function of protein content, and an acid-base indicator was used to denote the boundary displacement. As a proof of concept, standard model proteins and biological samples were chosen for the experiments to study the feasibility of the developed method. The experiments revealed that good linear calibration functions between V(MRB) and protein content (correlation coefficients R>0.98). The experiments further demonstrated the following merits of developed method: (1) weak influence of non-protein nitrogen additives (e.g., melamine) adulterated in protein samples, (2) good agreement with the classic Kjeldahl method (R=0.9945), (3) fast measuring speed in total protein analysis of large samples from the same source, and (4) low limit of detection (0.02-0.15 mg mL(-1) for protein content), good precision (R.S.D. of intra-day less than 1.7% and inter-day less than 2.7%), and high recoveries (105-107%). Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  2. Detection of Egg Production of Tegal Duck by Blood Protein Polymorphism

    Directory of Open Access Journals (Sweden)

    Ismoyowati Ismoyowati

    2008-05-01

    Full Text Available The aim of this research was to study the effect of transfferine, albumine, and haemoglobine loci to egg production characteristic of Tegal duck.  100 lying of Tegal ducks keeping by batteray-pen were used in this study.  Individual egg production was recorded until period of 120 days. Blood protein polymorphism analysed by electrophoresis method, and blood sample taken from each ducks.. Egg production and transfferine albumine, and haemoglobine phenotipe on electrophoresis gel were observed in this study.  Genotipe and gene frequencies and genetic variant were applied in data analysis. The result showed that (1 in the transferine locus were identified 3 aleles forming 4 genotipes (TfAA,TfAB, TfBB, and TfBC, (2 in albumine were identified 3 aleles forming 5 genotipes (AlbAA, AlbAB, AlbAC, AlbBB and AlbBC and (3 haemoglobine locus were identified 6 aleles forming 4 genotipes ((HbAA, HbAB, HbAC, HbBB, HbBC dan HbCC.  This study demostrated that B gene frequenci in transfferine, albumine and haemoglonine loci was highest than A and C gene frequency.  Tegal Duck with AA genotipe on all loci had higher egg production than BB and CC homozigote.  This research revealed that the most efective of selection method by haemoglobine protein polymorphism. (Animal Production 10(2: 122-128 (2008   Key Words: Tegal duck, egg production, selection, blood protein polymorphism

  3. Method for resolution and western blotting of very large proteins using agarose electrophoresis.

    Science.gov (United States)

    Greaser, Marion L; Warren, Chad M

    2015-01-01

    Proteins larger than 200 kDa are difficult to separate electrophoretically using polyacrylamide gels, and their transfer during western blotting is typically incomplete. A vertical SDS agarose gel system was developed that has vastly improved resolving power for very large proteins. Complete transfer of proteins as large as titin (Mr 3,000-3,700 kDa) onto blots can be achieved. The addition of a sulfhydryl reducing agent in the upper reservoir buffer and transfer buffer markedly improves the blotting of large proteins.

  4. Capillary electrophoresis separation of neutral organic compounds, pharmaceutical drugs, proteins and peptides, enantiomers, and anions

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Wei -Liang [Iowa State Univ., Ames, IA (United States)

    1999-02-12

    Addition of a novel anionic surfactant, namely lauryl polyoxyethylene sulfate, to an aqueous-acetonitrile electrolyte makes it possible to separate nonionic organic compounds by capillary electrophoresis. Separation is based on differences in the association between analytes and the surfactant. Highly hydrophobic compounds such as polyaromatic hydrocarbons are well separated by this new surfactant. Migration times of analytes can be readily changed over an unusually large range by varying the additive concentration and the proportion of acetonitrile in the electrolyte. Several examples are given, including the separation of four methylbenz[a]anthracene isomers and the separation of normal and deuterated acetophenone. The effect of adding this new surfactant to the acidic electrolyte was also investigated. Incorporation of cetyltrimethylammonium bromide in the electrolyte is shown to dynamically coat the capillary and reverse electroosmotic flow. Chiral recognition mechanism is studied using novel synthetic surfactants as chiral selectors, which are made from amino acids reacting with alkyl chloroformates. A satisfactory separation of both inorganic and organic anions is obtained using electrolyte solutions as high as 5 M sodium chloride using direct photometric detection. The effect of various salts on electrophoretic and electroosmotic mobility is further discussed. Several examples are given under high-salt conditions.

  5. Investigation and Comparison of Leishmania major Promastigote and Amastigote Protein Content by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    S. Soleimanifard

    2013-04-01

    Full Text Available ntroduction & Objective: Leishmania is a protozoan of the trypanosomatidae family. This pro-tozoan has two stages in its life cycle, promastigote form in sand flies and amastigote form in macrophage of mammalian hosts. The purpose of this study was identification and compari-son of proteins of Leishmania amastigote and promastigote stages. Materials & Methods: The present study is a cross sectional study of two forms of Leishmania major. To culture promastigotes , L.major (MRHO/IR/75/ER from previously infected Balb/c mice was transferred to modified N.N.N medium with overlay of liquid BHI and then transferred to RPMI-1640 at 26oc ± 1 for mass production. After isolation and growth, pro-mastigotes were transferred to liquid cell culture medium RPMI-1640 with pH 5.5 and incu-bated at 5% CO2 at 37oc for 72 hours until promastigote to amastigote transformation. Elec-trophoresis was performed with SDS-PAGE method to find and compare the molecular weight of the antigens of two stages. Results: The molecular weights of the bands observed in both forms were as follows: 19, 36, 50, 63, 65, 80, 90, 94, 96, 110- 130 KDa. The proteins in the surface of only promastigote were 22, 28 and 46 KDa and special proteins in the surface of amastigote were 12 and 32 KDa. Conclusion : According to this study Leishmania parasite has stage specific proteins. Various studies have shown that axenic amastigotes and tissue amastigotes are similar in their protein content. Therefore, based on stage specific proteins ,effective drugs and vaccines can be de-signed against leishmaniasis. (Sci J Hamadan Univ Med Sci 2013; 20 (1:1-8

  6. Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins.

    Science.gov (United States)

    Oh-Ishi, M; Satoh, M; Maeda, T

    2000-05-01

    A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.

  7. OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins.

    Science.gov (United States)

    Mena, Ma Luz; Moreno-Gordaliza, Estefanía; Moraleja, Irene; Cañas, Benito; Gómez-Gómez, Ma Milagros

    2011-03-04

    In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

    Science.gov (United States)

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide. PMID:26865351

  9. Effects of interferon gamma on Chlamydia trachomatis serovar A and L2 protein expression investigated by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, A; Christiansen, Gunna; Birkelund, Svend

    1999-01-01

    ]methionine and two-dimensional gel electrophoresis with immobilized pH gradients in order to investigate changes in the protein expression of C. trachomatis serovar A and L2 caused by treatment with IFN-gamma. In contrast to what was observed in C. trachomatis L2, our results showed that, in C. trachomatis A, down...

  10. Comparison of Yeast Cell Protein Solubilization Procedures for Two-dimensional Electrophoresis

    DEFF Research Database (Denmark)

    Harder, A; Wildgruber, R; Nawrocki, A

    1999-01-01

    with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea...... sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii)....... molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins...

  11. Identification of salivary gland proteins depleted after blood feeding in the malaria vector Anopheles campestris-like mosquitoes (Diptera: Culicidae.

    Directory of Open Access Journals (Sweden)

    Sriwatapron Sor-suwan

    Full Text Available Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles campestris-like, a potential malaria vector of Plasmodium vivax in Thailand, two-dimensional gel electrophoresis and nano-liquid chromatography-mass spectrometry techniques were used. Results showed that 19 major proteins were significantly depleted in three to four day-old mosquitoes fed on a first blood meal. For the mosquitoes fed the second blood meal on day 14 after the first blood meal, 14 major proteins were significantly decreased in amount. The significantly depleted proteins in both groups included apyrase, 5'-nucleotidase/apyrase, D7, D7-related 1, short form D7r1, gSG6, anti-platelet protein, serine/threonine-protein kinase rio3, putative sil1, cyclophilin A, hypothetical protein Phum_PHUM512530, AGAP007618-PA, and two non-significant hit proteins. To our knowledge, this study presents for the first time the salivary gland proteins that are involved in the second blood feeding on the day corresponding to the transmission period of the sporozoites to new mammalian hosts. This information serves as a basis for future work concerning the possible role of these proteins in the parasite transmission and the physiological processes that occur during the blood feeding.

  12. Identification of salivary gland proteins depleted after blood feeding in the malaria vector Anopheles campestris-like mosquitoes (Diptera: Culicidae).

    Science.gov (United States)

    Sor-suwan, Sriwatapron; Jariyapan, Narissara; Roytrakul, Sittiruk; Paemanee, Atchara; Phumee, Atchara; Phattanawiboon, Benjarat; Intakhan, Nuchpicha; Chanmol, Wetpisit; Bates, Paul A; Saeung, Atiporn; Choochote, Wej

    2014-01-01

    Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles campestris-like, a potential malaria vector of Plasmodium vivax in Thailand, two-dimensional gel electrophoresis and nano-liquid chromatography-mass spectrometry techniques were used. Results showed that 19 major proteins were significantly depleted in three to four day-old mosquitoes fed on a first blood meal. For the mosquitoes fed the second blood meal on day 14 after the first blood meal, 14 major proteins were significantly decreased in amount. The significantly depleted proteins in both groups included apyrase, 5'-nucleotidase/apyrase, D7, D7-related 1, short form D7r1, gSG6, anti-platelet protein, serine/threonine-protein kinase rio3, putative sil1, cyclophilin A, hypothetical protein Phum_PHUM512530, AGAP007618-PA, and two non-significant hit proteins. To our knowledge, this study presents for the first time the salivary gland proteins that are involved in the second blood feeding on the day corresponding to the transmission period of the sporozoites to new mammalian hosts. This information serves as a basis for future work concerning the possible role of these proteins in the parasite transmission and the physiological processes that occur during the blood feeding.

  13. Optimizing soluble protein extraction and two-dimensional polyacrylamide gel electrophoresis quality for extremophile ciliates.

    Science.gov (United States)

    Fulgentini, Lorenzo; Marangoni, Roberto; Colombetti, Giuliano

    2008-06-01

    An efficient protein extraction methodology is quite important for sample preparation and subsequent 2-D PAGE and MS analysis. Cell lysis is the first step in protein extraction and purification. Many techniques are available for cell disruption, including physical and detergent-based methods. Here, we report on a very fast and efficient detergent-free Tris-based method to extract the soluble fraction proteins of extremophile ciliates, comparing it with a detergent-based protocol. This comparison has been carried out by means of 2-D PAGE and subsequent MALDI-compatible silver staining of protein samples obtained from the intensely pigmented hypersaline ciliate Fabrea salina and the Antarctic hypotrich ciliate Euplotes focardii. Our results indicate that this fast and easy extraction method allows to obtain more clear crude extracts and more spot-abundant polyacrylamide gels.

  14. Analyzing modifiers of protein aggregation in C. elegans by native agarose gel electrophoresis

    NARCIS (Netherlands)

    Holmberg, Mats; Nollen, Ellen A A; Hatters, Danny M.; Hannan, Anthony J.

    2013-01-01

    The accumulation of specific aggregation-prone proteins during aging is thought to be involved in several diseases, most notably Alzheimer's and Parkinson's disease as well as polyglutamine expansion disorders such as Huntington's disease. Caenorhabditis elegans disease models with transgenic

  15. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    Science.gov (United States)

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry.

    Science.gov (United States)

    Fernández-Boo, S; Chicano-Gálvez, E; Alhama, J; Barea, J L; Villalba, A; Cao, A

    2014-05-01

    The genus Perkinsus includes protozoan parasites of a wide range of marine molluscs worldwide, some of which have been responsible for heavy mollusc mortalities and dramatic economic losses. This study was performed with the aim of increasing the knowledge of Perkinsus spp. proteome. Proteins extracted from in vitro cultured cells of three species of this genus, P. marinus, P. olseni and P. chesapeaki, were analysed using 2D electrophoresis. Four gels from each species were produced. Qualitative and quantitative comparisons among gels were performed with Proteamweaver software. Cluster analysis grouped the four gels of each Perkinsus sp.; furthermore, P. marinus and P. olseni gels were grouped in a cluster different from P. chesapeaki. Around 2000 spots of each species were considered, from which 213 spots were common to the 3 species; P. chesapeaki and P. marinus shared 310 spots, P. chesapeaki and P. olseni shared 315 spots and P. marinus and P. olseni shared 242 spots. A number of spots were exclusive of each Perkinsus species: 1161 spots were exclusive of P. chesapeaki, 1124 of P. olseni and 895 of P. marinus. A total of 84 spots, including common and species-specific ones, were excised from the gels and analysed using MALDI-TOF and nESI-IT (MS/MS) techniques. Forty-two spots were successfully sequenced, from which 28 were annotated, most of them clustered into electron transport, oxidative stress and detoxification, protein synthesis, carbohydrate metabolism, signal transduction, metabolic process and proteolysis. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Profiling of normal human leg lymph proteins using the 2-D electrophoresis and SELDI-TOF mass spectrophotometry approach.

    Science.gov (United States)

    Interewicz, B; Olszewski, W L; Leak, L V; Petricoin, E F; Liotta, L A

    2004-06-01

    The parenchymatous cells are supplied by nutrients transported in fluid from blood across the capillary wall. This fluid, called tissue fluid (TF), contains proteins originating from plasma as well as those synthesized and secreted by tissue cells. The protein composition of TF remains largely unknown. The TF which has entered lymphatics is called lymph (L). Harvesting L and measuring its proteins concentrations and identifying them provide an insight into biochemical processes in the TF. Here we describe our initial evaluation of the normal human prenodal L protein profile of m.w. 2.5 to 12.5 kDa using the ProteinChip SELDI MS system and compare it with that of plasma (P) protein. This is the first study in the literature providing evidence for the presence of the so far non-identified proteins in L as well as proteins identified in L but absent from P and conversely present in P but not in L. Evident differences between paired L and P samples have been found, along with similarities. Thirteen proteins were detected in P and seven in L in the region of 2.5 to 12.5 kDa. Five identical proteins, although of different relative intensity, were found in L and P. The proteins specific for L but not P had 7070 and 8619 ion values. P proteins absent from L were of 3890, 3969, 4078, 6863, 7676, 7778, 7847 and 7937 ion values. In addition to detecting some so far unknown proteins in L, these preliminary findings throw a new light on our understanding of the mechanism of transcapillary transport of low m.w. proteins. They challenge the commonly accepted notion of unlimited free diffusion of peptides across the capillary membrane.

  18. CAPILLARY ZONE ELECTROPHORESIS IONSPRAY MASS-SPECTROMETRY OF A SYNTHETIC DRUG PROTEIN CONJUGATE MIXTURE

    NARCIS (Netherlands)

    KOSTIAINEN, R; FRANSSEN, EJF; BRUINS, AP

    1993-01-01

    Low-molecular-mass proteins, such as lysozyme, may be suitable carriers to target drugs to the kidney. Naproxen, an anti-inflammatory drug, has been conjugated with lysozyme via a covalent amide bond formed between the carboxylic acid function of naproxen and the amino group of one of the lysines in

  19. Pyrenebutanoate as a dynamic protein modifier for fluorometric detection in capillary zone electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Horká, Marie; Šlais, Karel

    2002-01-01

    Roč. 23, 7-8 (2002), s. 1090-1095 ISSN 0173-0835 R&D Projects: GA AV ČR IAA4031901 Institutional research plan: CEZ:AV0Z4031919 Keywords : pyrenebutanoate * dynamic protein modifier * CZE Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.325, year: 2002

  20. Determination of lysergic acid diethylamide (LSD) in mouse blood by capillary electrophoresis/ fluorescence spectroscopy with sweeping techniques in micellar electrokinetic chromatography.

    Science.gov (United States)

    Fang, Ching; Liu, Ju-Tsung; Chou, Shiu-Huey; Lin, Cheng-Huang

    2003-03-01

    The separation and on-line concentration of lysergic acid diethylamide (LSD) in mouse blood was achieved by means of capillary electrophoresis/fluorescence spectroscopy using sodium dodecyl sulfate (SDS) as the surfactant. Techniques involving on-line sample concentration, including sweeping micellar electrokinetic chromatography (sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were applied; the optimum on-line concentration and separation conditions were determined. In the analysis of an actual sample, LSD was found in a blood sample from a test mouse (0.1 mg LSD fed to a 20 g mouse; approximately 1/10 to the value of LD(50)). As a result, 120 and 30 ng/mL of LSD was detected at 20 and 60 min, respectively, after ingestion of the doses.

  1. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

    International Nuclear Information System (INIS)

    Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y.

    1989-01-01

    The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [ 3 H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 ± 0.017% of hemoglobin in blood of normal adult, 0.11 ± 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [ 3 H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups

  2. Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting

    DEFF Research Database (Denmark)

    Bini, L; Sanchez-Campillo, M; Santucci, A

    1996-01-01

    Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two-dimensional gel electrophoresis on nonlinear wide-range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly...... reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N-terminal amino acid sequencing, we established the map positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein-rich outer membrane protein...... (OMP2), the DnaK-like, GroEL-like, and macrophage infectivity potentiator (MIP)-like proteins, the plasmid-encoded pgp3 protein, two ribosomal proteins (S1 and L7/L12), and the protein-elongation factor EF-Tu. Other proteins, for which gene assignment was not possible, have been identified by three...

  3. Renaturation of telomere-binding proteins after the fractionation by SDS-polyacrylamide gel electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Rotková, Gabriela

    2007-01-01

    Roč. 53, č. 7 (2007), s. 317-320 ISSN 1214-1178 R&D Projects: GA ČR(CZ) GA521/05/0055; GA AV ČR(CZ) IAA600040505; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : renaturation * telomere-binding proteins * telomeres Subject RIV: BO - Biophysics

  4. A stable and convenient protein electrophoresis titration device with bubble removing system.

    Science.gov (United States)

    Zhang, Qiang; Fan, Liu-Yin; Li, Wen-Lin; Cong, Feng-Song; Zhong, Ran; Chen, Jing-Jing; He, Yu-Chen; Xiao, Hua; Cao, Cheng-Xi

    2017-07-01

    Moving reaction boundary titration (MRBT) has a potential application to immunoassay and protein content analysis with high selectivity. However, air bubbles often impair the accuracy of MRBT, and the leakage of electrolyte greatly decreases the safety and convenience of electrophoretic titration. Addressing these two issues a reliable MRBT device with modified electrolyte chamber of protein titration was designed. Multiphysics computer simulation was conducted for optimization according to two-phase flow. The single chamber was made of two perpendicular cylinders with different diameters. After placing electrophoretic tube, the resident air in the junction next to the gel could be eliminated by a simple fast electrolyte flow. Removing the electrophoretic tube automatically prevented electrolyte leakage at the junction due to the gravity-induced negative pressure within the chamber. Moreover, the numerical simulation and experiments showed that the improved MRBT device has following advantages: (i) easy and rapid setup of electrophoretic tube within 20 s; (ii) simple and quick bubble dissipates from the chamber of titration within 2 s; (iii) no electrolyte leakage from the two chambers: and (iv) accurate protein titration and safe instrumental operation. The developed technique and apparatus greatly improves the performance of the previous MRBT device, and providing a new route toward practical application. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Genetic Variation through Polymorphism of Blood and Egg White Protein in Three Kinds of Kedu Chickens at Laying Period

    Directory of Open Access Journals (Sweden)

    LD Mahfudz

    2011-05-01

    Full Text Available Abstract. Genetic variation of five blood protein loci and three egg white protein loci in three kinds of Kedu chicken, namely the black skin and black feather (BB, black skin and white feather (BW and white skin and white feather (WW were investigated using polyacrilamide gel electrophoresis. The result showed that five blood loci (pre-albumin, albumin, post-transferin, transferin and hemoglobin and three egg white loci (lysozim, ovalbumin and conalbumin were found to be polymorphic in three kinds of Kedu chicken. BW has average higher heterozygosity than BB and WW. The result was shown in blood and egg white protein polymorphism. There was no difference on blood and egg white protein in the genetic variation. The average of heterozygosity of Kedu chicken of high production and low production was not different. Genetic distance among three populations of Kedu chicken showed that Kedu chicken BW was closer to WW than to BB. Key Words: Kedu chicken, protein polymorphism, blood, egg white, genetic variation

  6. Development of an SDS-gel electrophoresis method on SU-8 microchips for protein separation with LIF detection: Application to the analysis of whey proteins.

    Science.gov (United States)

    Del Mar Barrios-Romero, Maria; Crevillén, Agustín G; Diez-Masa, José Carlos

    2013-08-01

    This work describes the development of an SDS-gel electrophoresis method for the analysis of major whey proteins (α-lactalbumin, β-lactoglobulin, and BSA) carried out in SU-8 microchips. The method uses a low-viscosity solution of dextran as a sieving polymer. A commercial coating agent (EOTrol LN) was added to the separation buffer to control the EOF of the chips. The potential of this coating agent to prevent protein adsorption on the walls of the SU-8 channels was also evaluated. Additionally, the fluorescence background of the SU-8 material was studied to improve the sensitivity of the method. By selecting an excitation wavelength of 532 nm at which the background fluorescence remains low and by replacing the mercury arc lamp by a laser in the detection system, an LOD in the nanomolar range was achieved for proteins derivatized with the fluorogenic reagent Chromeo P540. Finally, the method was applied to the analysis of milk samples, demonstrating the potential of SU-8 microchips for the analysis of proteins in complex food samples. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Oligoclonal Pattern/Abnormal Protein Bands in Post-Treatment Plasma Cell Myeloma Patients: Implications for Protein Electrophoresis and Serum Free Light Chain Assay Results.

    Science.gov (United States)

    Singh, Gurmukh

    2017-08-01

    The impact of autologous stem cell transplantation (ASCT) in plasma cell myeloma patients on the frequency, quality, and timing of oligoclonal pattern in serum protein electrophoresis/immunofixation electrophoresis (SPEP/SIFE) and serum free light chain assay (SFLCA) was evaluated. Laboratory results and clinical data for 251 patients with plasma cell myeloma, who had SPEP/SIFE and/or SFLCA performed between January 2010 and December 2016 were reviewed. The results for SPEP/SIFE and SFLCA were compared in patients with ASCT to those without ASCT. The implications of oligoclonal pattern in interpretation of SPEP/SIFE and SFLCA - κ/λ ratio were addressed. In 251 patients, a total of 3,134 observations, of either SPEP/SIFE and/or SFLCA, were reviewed. One hundred fifty-nine patients received ASCT. The incidence of oligoclonal patterns was significantly higher after ASCT. More than half of the oligoclonal patterns developed in the first year after transplantation. In 13 of the 84 patients with lambda chain restricted plasma cell myeloma, the κ/λ ratio was kappa dominant in the presence of oligoclonal pattern. There was no reversal of κ/λ ratio in patients with kappa chain restricted plasma cell myelomas. ASCT is associated with significantly higher incidence of oligoclonal patterns than with chemotherapy alone. The presence of oligoclonal patterns has the potential to interfere with the interpretation of SPEP/SIFE and ascertainment of complete remission. At a minimum, the oligoclonal pattern caused an incorrect kappa dominant κ/λ ratio in 15.5% of patients with lambda chain restricted plasma cell myeloma. If a similar rate were to be applied to the 167 kappa chain myeloma patients, about 26 of these would have displayed an erroneous kappa chain dominant κ/λ ratio. The presence of oligoclonal pattern further degrades the performance of already dubious SFLCA. The need for recording the location of monoclonal spike in SPEP/SIFE and higher resolution protein

  8. Continuous-Flow Electrophoresis of DNA and Proteins in a Two-Dimensional Capillary-Well Sieve.

    Science.gov (United States)

    Duan, Lian; Cao, Zhen; Yobas, Levent

    2017-09-19

    Continuous-flow electrophoresis of macromolecules is demonstrated using an integrated capillary-well sieve arranged into a two-dimensional anisotropic array on silicon. The periodic array features thousands of entropic barriers, each resulting from an abrupt interface between a 2 μm deep well (channel) and a 70 nm capillary. These entropic barriers owing to two-dimensional confinement within the capillaries are vastly steep in relation to those arising from slits featuring one-dimensional confinement. Thus, the sieving mechanisms can sustain relatively large electric field strengths over a relatively small array area. The sieve rapidly sorts anionic macromolecules, including DNA chains and proteins in native or denatured states, into distinct trajectories according to size or charge under electric field vectors orthogonally applied. The baseline separation is achieved in less than 1 min within a horizontal migration length of ∼1.5 mm. The capillaries are self-enclosed conduits in cylindrical profile featuring a uniform diameter and realized through an approach that avoids advanced patterning techniques. The approach exploits a thermal reflow of a layer of doped glass for shape transformation into cylindrical capillaries and for controllably shrinking the capillary diameter. Lastly, atomic layer deposition of alumina is introduced for the first time to fine-tune the capillary diameter as well as to neutralize the surface charge, thereby suppressing undesired electroosmotic flows.

  9. Blood chemistry, protein electrophoresis, and hematologic values of captive Spanish imperial eagles (Aquila adalberti).

    Science.gov (United States)

    García-Montijano, Marino; García, Alfonso; Lemus, Jesús A; Montesinos, Andrés; Canales, Rocío; Luaces, Inés; Pereira, Pablo

    2002-06-01

    Hemograms and plasma chemistry values are presented for six male and six female, adult, clinically normal, captive Spanish imperial eagles (Aquila adalberti). No value was substantially different from that which might be predicted on the basis of work in other related species. This data should prove useful for the interpretation of laboratory findings in future clinical cases of this endangered species of eagle.

  10. Chicken liver TGGCA protein purified by preparative mobility shift electrophoresis (PMSE) shows a 36.8 to 29.8 kd microheterogeneity.

    Science.gov (United States)

    Rupp, R A; Sippel, A E

    1987-12-10

    The TGGCA protein, the chicken homologue of HeLa cell NF-I, was purified to homogeneity from liver tissue by a procedure which includes preparative mobility shift electrophoresis (PMSE) as the final step. PMSE was here adjusted for the isolation of the TGGCA protein, but can be used as a general method to characterize the protein moiety of specific DNA-binding proteins. The TGGCA protein is a family of 6 protein species, which show minor differences in molecular weight from 36.8kd to 29.8kd. This microheterogeneity differs from the size distribution reported for HeLa cell NF-I polypeptides. All species of the TGGCA protein bind identically to a synthetic DNA-binding site and appear to be highly related in primary structure. We discuss the possible functional importance of this microheterogeneity.

  11. Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Ritu Rawal

    Full Text Available In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite

  12. GENETIC VARIATION IN THREE BREEDS OF INDONESIAN LOCAL DUCKS BASED ON BLOOD AND EGG WHITE PROTEIN POLYMORPHISM

    Directory of Open Access Journals (Sweden)

    S. Johari

    2014-10-01

    Full Text Available A study of genetic variation of four blood protein loci and three egg white loci in three breeds ofIndonesian local ducks (Magelang duck = MAD, Tegal duck = TED and Mojosari duck= MOD wasinvestigated by using polyacrilamide gel electrophoresis (PAGE. The result showed that four bloodprotein loci (Pre-albumin, Albumin, Transferrin and Post-transferrin and three egg white loci(Ovalbumin, Ovoglobulin and Conalbumin were found to be polymorphic in three breeds of localducks. There was no difference on blood and egg white protein polyimorphism. The average value ofheterozygosity on three breeds of local ducks had a genetic potential to determine the controlled matingsystem and selection to obtain pure line. The closest phylogenetic relationship among the three breeds oflocal ducks occurred between MAD and TED.

  13. Differences in alcohol-soluble protein from genetically altered wheat using capillary zone electrophoresis, one- and two-dimensional electrophoresis and a novel gluten matrix association factor analysis

    Science.gov (United States)

    Wheat protein composition and organization play interrelated roles in determining physical properties for technological purposes. In prior research, a number of isogenic wheat lines of Bobwhite that have high levels of expression of the native Dx5 and/or Dy10 high molecular weight subunits (HMW-GS)...

  14. Protein supplementation lowers blood pressure in overweight adults : effect of dietary proteins on blood pressure (PROPRES), a randomized trial

    NARCIS (Netherlands)

    Teunissen-Beekman, Karianna F. M.; Dopheide, Janneke; Geleijnse, Johanna M.; Bakker, Stephan J. L.; Brink, Elizabeth J.; de Leeuw, Peter W.; van Baak, Marleen A.

    Background: Dietary protein intake may help to manage blood pressure (BP) and prevent complications associated with elevated BR Objective: The objective of this study was to determine whether 4 wk of increased protein intake (similar to 25% compared with similar to 15% of energy intake that

  15. Protein supplementation lowers blood pressure in overweight adults: effect of dietary proteins on blood pressure (PROPRES), a randomized trail

    NARCIS (Netherlands)

    Teunissen-Beekman, K.F.M.; Dopheide, J.; Geleijnse, J.M.; Bakker, S.J.L.; Brink, E.J.; Leeuw, de P.W.; Baak, van M.A.

    2012-01-01

    Background: Dietary protein intake may help to manage blood pressure (BP) and prevent complications associated with elevated BP. Objective: The objective of this study was to determine whether 4 wk of increased protein intake (~25% compared with ~15% of energy intake that isoenergetically replaces

  16. Protein supplementation lowers blood pressure in overweight adults: Effect of dietary proteins on blood pressure (PROPRES), a randomized trial

    NARCIS (Netherlands)

    Teunissen-Beekman, K.F.M.; Dopheide, J.; Geleijnse, J.M.; Bakker, S.J.L.; Brink, E.J.; Leeuw, P.W. de; Baak, M.A. van

    2012-01-01

    Background: Dietary protein intake may help to manage blood pressure (BP) and prevent complications associated with elevated BP. Objective: The objective of this study was to determine whether 4 wk of increased protein intake (∼25% compared with ;15% of energy intake that isoenergetically replaces

  17. Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

    Science.gov (United States)

    Chambers, Andrew G.; Percy, Andrew J.; Hardie, Darryl B.; Borchers, Christoph H.

    2013-09-01

    Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77 %-92 % within replicates and the majority of these repeated proteins (70 %) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

  18. Genetic characteristic of swamp buffalo (Bubalus bubalis) from Pampangan, South Sumatra based on blood protein profile

    Science.gov (United States)

    Windusari, Yuanita; Hanum, Laila; Wahyudi, Rizki

    2017-11-01

    Swamp buffalo (Bubalus bubalis) is an endemic species and one of the genetic wealth of South Sumatra with a distribution area in the district of Pampangan (OganIlir and OganOganIlir). Suspected inbreeding causes decreased phenotypic properties. Inbreeding among various swamp buffalo is certainly not only lower the qualities but also genotypes and phenotypes. It is of interest to determine kinship variants swamp buffaloes from Pampangan through the analysis of a blood protein profile. Blood protein profile of four variants swamps buffalo was studied by using five electrophoresis system i.e. pre-albumin (Palb), albumin (Alb), ceruloplasmin (Cp), transferrin (Tf) and transferrin post (Ptf). In this paper, it is obtained that there was no significant differences among the four variants of the buffaloes were used as a sample. Prealbumin has two alleles (Palb1 and Palb2), albumin has three alleles (Alba, AlbB, AlbC), ceruloplasmin has one allele (BPA), post-transferrin has one allele (PTFA) with an allele frequency 1.0000 at any time transferrin has two alleles (TFA and TFB) with the allele frequency of 0.7500 and 1.0000. Characteristics prealbumin (Palb), albumin (Alb), ceruloplasmin (Cp), and post-transferrin (P-tf) is monomorphic, while transferrin is polymorphic average heterozygosity values all loci (H) 0.1286. Based on average heterozygosity, the swamp buffalo (Bubalusbubalis) from Pampangan has low genetic variation and closest genetic relationship.

  19. High pressure electrophoresis in narrow bore glass tubes: One- and two-dimensional separations of protein subunits

    Science.gov (United States)

    Erijman, Leonardo; Clegg, Robert M.

    1996-03-01

    A mini-gel tube electrophoresis apparatus that is easily constructed and simple to operate has been developed. The system can be accommodated in standard commercially available high pressure tubing, and has been tested at up to 200 MPa. The narrow diameter of the glass tubes allows rapid and efficient dissipation of heat. Adequate buffer capacity is maintained in the low volume anode reservoir by increasing the concentration of the buffer. Analytical separations can be achieved in short times with high resolution. After the electrophoresis has been carried out at elevated pressure, the gel can easily be extruded from the tube and loaded onto a standard slab gel for a second-dimensional run at atmospheric pressure. We illustrate the application of this apparatus with the high pressure gel electrophoresis separation and subsequent identification of the constituent subunits of E. coli RNA polymerase.

  20. INFLUENCE OF NATURAL IMMUNOMODULATORS ON PROTEIN FRACTIONS AND CORTISOL CONTENT IN RABBIT BLOOD UNDER STRESS

    Directory of Open Access Journals (Sweden)

    Grabovskyi S.

    2015-08-01

    Full Text Available The results of determination of protein fractions, cortisol content in blood of rabbits, which further added to the feed of natural origin biologically active substances are presented in the article. As an antistressors and immunomodulators in pre-slaughter period are using of spleen extract biologically active substances were obtained with ultrasound application. The purpose of research — determination of changes of protein fractions, cortisol content in rabbits blood before slaughter and their correction of natural origin biologically active substances (spleen extract. Object and research methods. The experiment was conducted on 15 rabbits with standard diet. Three groups of rabbits five month of age (5 rabbits each was formed for research. The spleen extract were using as an biologically active substances to the feed rabbits in pre-slaughter period (five days before slaughter. The extracts were applied to feed by aerosol method (70 °alcohol solution of spleen extract volume of 1.4 ml per rabbit (group I. The rabbits (group II received to the feed in the same way of 70 °alcohol solution in the same volume. The control group rabbits received the standard feed in the same volume. The feed eating by rabbits was exercised daily. The rabbits ate food completely. The rabbits slaughter was carried out in the morning. The blood plasma protein fractions separation was carried out by horizontal electrophoresis in polyacrylamide gel (PAAG. Mathematical treatment of the research results worked statistically using the software package Statistica 6.0 and Microsoft Excel for Windows XP. Probability differences was assessed by Student t-test and results considered likely at P ≤ 0.05. Results and discussion. We measured the ratio of blood plasma protein fractions of rabbits, which in addition to the feed fed of natural origin biologically active substances. As a result of research was found that aerosol introduction of the spleen extract to the rabbits

  1. Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins

    Science.gov (United States)

    Liu, Zhen; Li, Ping; Bian, Weiwei; Yu, Jingkai; Zhan, Jinhua

    2016-05-01

    Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 103 times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state.

  2. Molecular interactions of graphene oxide with human blood plasma proteins

    Science.gov (United States)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  3. Studies on Some Biophysical Properties of the Serum Protein of Mice blood exposed to an electric field

    International Nuclear Information System (INIS)

    Hanafy, M.S.

    2005-01-01

    As an indication of the effect of the electric field on each of the dielectric properties and the molecular structure of the serum protein of the mice blood, an electric field of a 6 kv/m strength and 50 Hz frequency was directed to three groups of mice for exposure periods 30, 45 and 60 days respectively, and investigated directly. Another group was exposed to also 60 days, but investigated after 30 days from switching off the electric field for delayed effect studies. The molecular structure of the serum protein was studied by measuring each of the dielectric relaxation and the electric conductivity in the frequency range 0.15 MHz at 4 ± 0.5 degree C and the dielectric increment (Δ), relaxation time (τ) and average molecular radii (τ) were calculated for all groups. The absorption spectra of the extracted protein were also measured in the wavelength range 200 600 nm. Moreover, electrophoresis of enzymes B-esterase, lactate and Malate dehydrogenase extracted from the blood serum of exposed mice were taken by using the gel electrophoresis technique. The results indicated that exposure of the animals to 50 H, 6 kv/m electric field resulted in the decrease of serum protein permittivity values and increase its conductivity a fact that indicates pronounced changes in the molecular structure of total serum protein the exposed mice. In addition, the intensity of the absorption spectral bands of serum protein of exposed mice were found to decrease relative to unexposed mice. Also the enzymes B-esterase and lactate dehydrogenase were slightly affected by exposing to the electric field whereas their number of bands and their intensities changed relative to the unexposed mice but the malate dehydrogenase was not affected

  4. A method for studies on interactions between a gold-based drug and plasma proteins based on capillary electrophoresis with inductively coupled plasma mass spectrometry detection

    DEFF Research Database (Denmark)

    Nguyen, Tam T T N; Østergaard, Jesper; Gammelgaard, Bente

    2015-01-01

    An analytical method based on capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICP-MS) detection was developed for studies on the interaction of gold-containing drugs and plasma proteins using auranofin as example. A detection limit of 18 ng/mL of auranofin...... corresponding to 5.2 ng/mL Au and a precision of 1.5 % were obtained. Kinetic studies of the interaction between auranofin and protein were performed by incubation in aqueous solutions as well as 20 % human plasma at 37 °C. The reaction of auranofin with human serum albumin (HSA) and plasma proceeded fast; 50...... was the major auranofin-interacting protein in plasma. The CE-ICP-MS method is proposed as a novel approach for kinetic studies of the interactions between gold-based drugs and plasma proteins. Graphical Abstract Development of a CE-ICP-MS based method allows for studies on interaction of the gold containing...

  5. [Production of human proteins in the blood of transgenic animals

    NARCIS (Netherlands)

    Massoud, M.; Bischoff, Rainer; Dalemans, W.; Pointu, H.; Attal, J.; Schultz, H.; Clesse, D.; Stinnakre, M.G.; Pavirani, A.; Houdebine, L.M.

    1990-01-01

    The human alpha 1-antitrypsin gene has been microinjected into rabbit embryos. A line of transgenic rabbits has thus been established. Human alpha 1-antitrypsin was found in the blood of transgenic animals at the concentration of 1 mg/ml plasma. The human protein was active and separable from its

  6. In-capillary enrichment, proteolysis and separation using capillary electrophoresis with discontinuous buffers: application on proteins with moderately acidic and basic isoelectric points.

    Science.gov (United States)

    Nesbitt, Chandra A; Yeung, Ken K-C

    2009-01-01

    Advances in mass spectrometry and capillary-format separation continue to improve the sensitivity of protein analysis. Of equal importance is the miniaturization of sample pretreatment such as enrichment and proteolysis. In a previous report (Nesbitt et al., Electrophoresis, 2008, 29, 466-474), nanoliter-volume protein enrichment, tryptic digestion, and partial separation was demonstrated in capillary electrophoresis followed by MALDI mass spectral analysis. A discontinuous buffer system, consisting of ammonium (pH 10) and acetate (pH 4), was used to create a pH junction inside the capillary, trapping a protein with a neutral isoelectric point, myoglobin (pI 7.2). Moreover, co-enrichment of myoglobin with trypsin led to an in-capillary digestion. In this paper, the ability of this discontinuous buffer system to perform similar in-capillary sample pretreatment on proteins with moderately acidic and basic pI was studied and reported. Lentil lectin (pI 8.6) and a multi-phosphorylated protein, beta-casein (pI 5.1), were selected as model proteins. In addition to the previously shown tryptic digestion, proteolysis with endoproteinase Asp-N was also performed. Digestion of these acidic and basic pI proteins produced a few peptides with extreme pI values lying outside the trapping range of the discontinuous buffer. An alteration in the peptide trapping procedure was made to accommodate these analytes. Offline MALDI mass spectral analysis confirmed the presence of the expected peptides. The presented miniaturized sample pretreatment methodology was proven to be applicable on proteins with a moderately wide range of pI. Flexibility in the choice of protease was also evident.

  7. Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae

    Science.gov (United States)

    2013-01-01

    Background Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae. Results Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI > 7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to

  8. Analysis of methylene blue and its metabolites in blood by capillary electrophoresis/electrospray ionization mass spectrometry.

    Science.gov (United States)

    Yang, Fang; Xia, Shifei; Liu, Zhencai; Chen, Jian; Lin, Yonghui; Qiu, Bin; Chen, Guonan

    2011-03-01

    A method for the determination of methylene blue (MB) and its metabolites (azure A, azure B and azure C) in rat blood by CE-electrospray ionization mass spectrometry (CE-ESI-MS) was developed in this paper. Different analytical parameters were investigated in detail such as pH and concentration of separation buffer, and ESI-MS instrumental parameters. Under the optimum conditions, MB and its metabolites were separated and detected in 27.3 min. LODs (defined as S/N=3) of this method were 0.22, 0.25, 0.10 and 0.30 μg/mL for MB, azure A, azure B and azure C, respectively. To get a satisfactory extraction efficiency of MB and its metabolites in rat blood, different extraction solutions were studied. By using this method, MB and its metabolites (azure A, azure B and azure C) were successfully analyzed in rat blood samples. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Proteomic analysis of halotolerant proteins under high and low salt stress in Dunaliella salina using two-dimensional differential in-gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Yan-Long Jia

    2016-01-01

    Full Text Available Abstract Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE was used to investigate the expression of halotolerant proteins under high (3 M NaCl and low (0.75 M NaCl salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress.

  10. A neutral polyacrylate copolymer coating for surface modification of thiol-ene microchannels for improved performance of protein separation by microchip electrophoresis

    DEFF Research Database (Denmark)

    Mesbah, Kiarach; Mai, T.D.; Jensen, Thomas Glasdam

    2016-01-01

    We have investigated the behavior of thiol-ene substrates that is a class of promising materials for lab-on-a-chip electrophoresis applications. Two polymeric materials were prepared by copolymerization of N, N-dimethylacrylamide (DMA), (3-(methacryloyl-oxy)propyl)trimethoxysilane (PMA) and 3......-(DMA-PMAMAPS) copolymer were evaluated in terms of surface hydrophilicity, suppression and stability of electro-osmotic flow and prevention of protein adsorption. Surface modification of thiol-ene containing a 20 % excess of thiols with the terpolymer p-(DMA-PMA-MAPS) was found to offer the most stable coating and most...

  11. A subtle calculation method for nanoparticle’s molar extinction coefficient: The gift from discrete protein-nanoparticle system on agarose gel electrophoresis

    Science.gov (United States)

    Zhong, Ruibo; Yuan, Ming; Gao, Haiyang; Bai, Zhijun; Guo, Jun; Zhao, Xinmin; Zhang, Feng

    2016-03-01

    Discrete biomolecule-nanoparticle (NP) conjugates play paramount roles in nanofabrication, in which the key is to get the precise molar extinction coefficient of NPs. By making best use of the gift from a specific separation phenomenon of agarose gel electrophoresis (GE), amphiphilic polymer coated NP with exact number of bovine serum albumin (BSA) proteins can be extracted and further experimentally employed to precisely calculate the molar extinction coefficient of the NPs. This method could further benefit the evaluation and extraction of any other dual-component NP-containing bio-conjugates.

  12. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Madsen, P S; Hokland, M; Ellegaard, J

    1988-01-01

    We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific Mo......Abs, and separated to a high degree of homogeneity by FACS into CD4+ helper T cells, CD8+ suppressor T cells, CD20+ B cells, and N901 (NHK-1)+ NK cells. The four lymphocyte subpopulations were labeled with [35S]methionine for 14 hr, solubilized in lysis buffer, and analyzed by two-dimensional gel electrophoresis...... (IEF). Of about 1000 proteins resolved in each case, most were found to be common to all subpopulations. However, eight putative markers for B1+ (proteins 5525, Mr = 63,700; 5621, Mr = 63,700; 8311, Mr = 36,900; 2202, Mr = 36,300; 6121, Mr = 30,300; 106, Mr = 29,300; 5009, Mr = 23,000; 8012, Mr = 11...

  13. Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

    Science.gov (United States)

    Takeda, Kumiko; Tasai, Mariko; Akagi, Satoshi; Watanabe, Shinya; Oe, Mika; Chikuni, Koichi; Ohnishi-Kameyama, Mayumi; Hanada, Hirofumi; Nakamura, Yoshiaki; Tagami, Takahiro; Nirasawa, Keijiro

    2011-04-01

    Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P result implicates mitochondrial-related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals. Copyright © 2011 Wiley-Liss, Inc.

  14. Conjugation reactions in the preparations of quantum dot-based immunoluminescent probes for analysis of proteins by capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Voráčová, Ivona; Klepárník, Karel; Hezinová, Věra; Přikryl, Jan; Foret, František

    2011-01-01

    Roč. 400, č. 2 (2011), s. 369-379 ISSN 1618-2642 R&D Projects: GA ČR GA203/08/1680; GA MŠk LC06023; GA ČR GAP301/11/2055 Institutional research plan: CEZ:AV0Z40310501 Keywords : CdTe quantum dots * luminescent probes * capillary zone electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.778, year: 2011

  15. Identification of Increased Amounts of Eppin Protein Complex Components in Sperm Cells of Diabetic and Obese Individuals by Difference Gel Electrophoresis*

    Science.gov (United States)

    Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jürgen; Dittmar, Gunnar; Glander, Hans-Jürgen; Hoflack, Bernard; Kriegel, Thomas M.

    2011-01-01

    Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the

  16. Analysis of Protein Expression inBrucella abortusMutants with Different Growth Rates by Two Dimensional Gel Electrophoresis and LC-MS/MS Peptide Analysis.

    Science.gov (United States)

    Park, Woo Bin; Im, Young Bin; Soojin, Shim; Yoo, Han Sang

    2017-10-13

    Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonosis. Pathogenesis of the Brucella abortus infection is so complicates. Thus, several researches have made to solve mechanism in infection of Brucella abortus . While several proteins are revealed as the pathogenic factors by previous researches, underlying mechanism of the Brucella abortus infection is still left to be resolved. We identify the proteins showing the different expression level in Brucella abortus mutants with different biological characteristics that generated by random insertion of transposon. Five mutants were selected based on the biological characteristics, especially the growth features. Total proteins of mutants and wild type were purified and subjected to two dimensional gel electrophoresis. 30 spots of proteins were selected each with an increase and decrease, a change of more than 2-fold was confirmed as compared with the wild type. Selected spots were analyzed using LC-MS/MS peptide analysis. DnaK and ClpB involved in protein aggregation were simultaneously increased. Proteins associated with energy metabolism, SecA and GAPDH were reduced in the expression in some mutants that slower growth rates than wild type. Mutants with slower growth showed decrease with energy metabolism related proteins, while mutants with faster growth showed increase in the pathogenicity related proteins.

  17. Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins.

    Science.gov (United States)

    Zhang, Chao-Xuan; Meagher, Michael M

    2017-03-21

    Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.

  18. Protein/RNA coextraction and small two-dimensional polyacrylamide gel electrophoresis for proteomic/gene expression analysis of renal cancer biopsies.

    Science.gov (United States)

    Barbero, Giovanna; Carta, Franco; Giribaldi, Giuliana; Mandili, Giorgia; Crobu, Salvatore; Ceruti, Carlo; Fontana, Dario; Destefanis, Paolo; Turrini, Francesco

    2006-02-01

    A small amount of bioptic tissue ( approximately 5-10mg of fresh tissue) usually does not contain enough material to extract protein and RNA separately, to obtain preparative two-dimensional polyacrylamide gel electrophoresis (2-DE), and to identify a large number of separated proteins by MS. We tested a method, on small renal cancer specimens, for the coextraction of protein and RNA coupled with 2-DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or quadrupole time-of-flight (Q-TOF) analysis. We coextracted 0.28+/-0.05mg of proteins and 2.5+/-0.33microg of RNA for each 10mg of renal carcinoma tissue. Small and large 2-DE gels were compared: they showed a similar number of spots, and it was possible to match each other; using small format gels, one-fifth of the protein amount was required to identify, by Q-TOF analysis, the same number of proteins identifiable in large-format gel using MALDI-TOF analysis. Quality of RNA coextracted with the proteins was tested by real-time PCR on a set of housekeeping genes. They were quantified with high amplification efficiency and specificity. In conclusion, using 5 to 10mg of fresh tissue, it was possible to perform comprehensive parallel proteomic and genomic analysis by high-resolution, small-format 2-DE gels, allowing approximately 300 proteins identification and 1000 genes expression analysis.

  19. Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

    Directory of Open Access Journals (Sweden)

    Zeng An-Ping

    2003-12-01

    Full Text Available Abstract Separation of proteins by two-dimensional gel electrophoresis (2-DE coupled with identification of proteins through peptide mass fingerprinting (PMF by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.

  20. Computer-assisted detection of monoclonal components: results from the multicenter study for the evaluation of CASPER (Computer Assisted Serum Protein Electrophoresis Recognizer) algorithm.

    Science.gov (United States)

    Ognibene, Agostino; Graziani, Maria S; Caldini, Anna; Terreni, Alessandro; Righetti, Gabriella; Varagnolo, Maria C; Campanella, Ada; Martelli, Marinella; Mancini, Rita; Rizzotti, Paolo; Plebani, Mario; Mori, Marco; Gaspari, Giovanni; Motta, Roberto; Galli, Gianni; Fabris, Massimiliano; Messeri, Gianni

    2008-01-01

    To investigate the potential use of Artificial Neural Network (ANN) in the evaluation of serum protein electrophoresis, we set up a multicenter study involving six Italian laboratories. For this purpose, we developed an algorithm named CASPER (Computer Assisted Serum Protein Electrophoresis Recognizer). A total of 59,516 samples from the six centers were divided into three groups. Training and validation sets were used to develop the neural network, whereas evaluation set was used to test the performance of CASPER in recognizing abnormal electrophoretic profiles. CASPER showed 93.0% sensitivity and 47.4% specificity. CASPER sensitivity and specificity ranged in the six sites from 88% (site 3) to 97% (site 5) and from 36% (site 6) to 53% (site 3), respectively. Sensitivity for gamma zone was 94.6%, for beta zone 89.7% and for oligoclonal patterns 92.0%. The sensitivity of the CASPER algorithm does not allow us to recommend its use as a replacement for the visual inspection, but it could be helpful in avoiding accidental misclassifications by the operator. Moreover, the CASPER algorithm may be a useful tool for training operators and students. This study evidenced a high inter-observer variability, which should be addressed in a dedicated study. Data set to train and validate ANNs should contain a huge range and an adequate number of different abnormalities.

  1. [Biosensors as a tool to use in the experiments on blood and selected blood proteins].

    Science.gov (United States)

    Pawlaczyk, Izabela; Ziewiecki, Rafał; Czerchawski, Leszek; Krotkiewski, Hubert; Gancarz, Roman

    2013-01-01

    Over last few years, biosensors have become increasingly used as a research tool. Using innovative techniques of detection and estimation of the strength of intermolecular bonds, is particularly important in biochemical processes, including the study of mechanisms of interactions between plasma proteins in the circulatory system. With the numerous enhancements biosensors have become extremely sensitive devices, allowing for analysis of diverse biological material. Moreover, the use of immobilization techniques enables to use sample repeatedly, which significantly reduces costs and the ability to monitor tests in real-time shorten the time of experiment. The presented work discusses examples of the usage of biosensors in the research on the mechanisms of the interactions of blood plasma proteins. The experiments on cancer biomarkers present in the blood circulation system, protein C deficiency causing non-controlled hemorrhagic accidents, and on the level of fibrinogen, as well as 20S proteasome concentration in plasma, are just some examples of biosensors usage in the analyses of blood. They are also applicable in the research work performed the project WROVASC--Integrated Cardiovascular Center, concerning the mechanisms of anticoagulant activity in blood plasma of the polyphenolic-polysaccharide macromolecules of plant origin.

  2. Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

    Directory of Open Access Journals (Sweden)

    Mignogna Giuseppina

    2006-03-01

    Full Text Available Abstract In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively.

  3. Acetonitrile as a buffer additive for free zone capillary electrophoresis separation and characterization of maize (Zeamays L. ) and sorghum (Sorghum bicolor L. Moench) storage proteins.

    Science.gov (United States)

    Bean, S R; Lookhart, G L; Bietz, J A

    2000-02-01

    An improved method for separating and characterizing maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) storage proteins by free zone capillary electrophoresis (FZCE) was developed. Previous electrophoretic methods for analyzing these proteins required high concentrations of urea to maintain protein solubility during separation. To overcome disadvantages of urea, we developed a FZCE method that mimicked reversed-phase high-performance liquid chromatography (RP-HPLC) in that it used high levels of acetonitrile (ACN) at low pH. The optimized FZCE buffer system consisted of 80 mM phosphate-glycine buffer, nominal pH 2.5, containing 60% ACN and a cellulose derivative to dynamically coat capillary walls. Resolution was similar to or higher than that previously achieved by FZCE buffers utilizing 8 M urea as a buffer additive. ACN concentrations of at least 50% were necessary to achieve acceptable separations; this ACN concentration is approximately that necessary to extract these storage proteins. ACN was equally effective as traditional ethanol solvents and 8 M urea for solubilizing maize and sorghum proteins. The ACN-based FZCE buffer system gave high repeatability (buffers. This FZCE method may be applicable for the analysis of other hydrophobic proteins without the use of urea.

  4. Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

    Science.gov (United States)

    Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

    2010-09-15

    The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  5. Quantification of Whey Protein Content in Infant Formulas by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE): Single-Laboratory Validation, First Action 2016.15.

    Science.gov (United States)

    Feng, Ping; Fuerer, Christophe; McMahon, Adrienne

    2017-03-01

    Protein separation by sodium dodecyl sulfate-capillary gel electrophoresis, followed by UV absorption at 220 nm, allows for the quantification of major proteins in raw milk. In processed dairy samples such as skim milk powder (SMP) and infant formulas, signals from individual proteins are less resolved, but caseins still migrate as one family between two groups of whey proteins. In the first group, α-lactalbumin and β-lactoglobulin migrate as two distinct peaks. Lactosylated adducts show delayed migration times and interfere with peak separation, but both native and modified forms as well as other low-MW whey proteins still elute before the caseins. The second group contains high-MW whey proteins (including bovine serum albumin, lactoferrin, and immunoglobulins) and elutes after the caseins. Caseins and whey proteins can thus be considered two distinct nonoverlapping families whose ratio can be established based on integrated areas without the need for a calibration curve. Because mass-to-area response factors for whey proteins and caseins are different, an area correction factor was determined from experimental measurement using SMP. Method performance assessed on five infant formulas showed RSDs of 0.2-1.2% (within day) and 0.5-1.1% (multiple days), with average recoveries between 97.4 and 106.4% of added whey protein. Forty-three different infant formulas and milk powders were analyzed. Of the 41 samples with manufacturer claims, the measured whey protein content was in close agreement with declared values, falling within 5% of the declared value in 76% of samples and within 10% in 95% of samples.

  6. Discrimination between peak spreading in capillary zone electrophoresis of proteins due to interaction with the capillary wall and due to protein microheterogeneity

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Radko, S. P.; Chrambach, A.

    2001-01-01

    Roč. 22, č. 1 (2001), s. 66-70 ISSN 0173-0835 Institutional research plan: CEZ:AV0Z4031919 Keywords : capillary electrophoresis * dispersion mechanism * wall interaction versus microheterogeneity Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.282, year: 2001

  7. [Comparative study of the oncornavirus A and D proteins of human J-96 cells by the methods of isoelectric focusing and polyacrylamide gel electrophoresis].

    Science.gov (United States)

    Kitsak, V Ia; Moĭsiadi, S A; Delimbetova, G A; Dotsenko, V L; Krispin, T I

    1978-01-01

    Three peaks of 14C-radioactivity with buoyant densities of 1.23--1.24, 1.26 and 1.29 g/ml were detected in a cytoplasmic extract of J-96 cells upon equilibrium centrifugation in sucrose gradient. Electron microscopy of the 1.23--1.24 g/ml buoyant density fraction revealed particles 60--80 nm in diameter showing morphology characteristic of oncornavirus A. Isoelectric focusing in polyacrylamide gel showed polypeptides of extracellular D virus and oncornavirus A to differ in isofocusing points (pI). Proteins of extracellular D virus were localized in zones with pH 3.7, 4.0, 4.4, 4.7, 5.6, 6.5, 8.1, 9.45, and 10.0; polypeptide of intracytoplasmic oncornavirus A had the following isofocusing points: 4.0, 4.9, 6.7, 7.3, 9.0, 9.45 and over 10.0. Electrophoresis of polypeptides of D virus and intracellular oncornavirus A revealed differences in the molecular weights of the components. No proteins with molecular weights of 10,000, 12,000, 15,000, and 27,000 dalton characteristic of the extracellular D virus were found in oncornavirus A virions. The analysis of protein patterns obtained in parallel experiments of isoelectric focusing and polyacrylamide gel electrophoresis suggests that oncornaviruses A and D of J-96 cells differ in the characteristics (pI and molecular weight) of the structural polypeptide components.

  8. Blood plasma proteins and protein fractions in roe deer Capreolus capreolus L.

    Directory of Open Access Journals (Sweden)

    Dorota CYGAN-SZCZEGIELNIAK

    2015-09-01

    Full Text Available The aim of the research was to investigate some selected biochemical blood parameters in roe deer (Capreolus capreolus L.. The experiment covered 15 from 2 to 3-year-old bucks from Kuyavian-Pomeranian Voivodeship. The animals were shot by individual hunters on the shooting grounds during the hunting season of 2008/2009 (in the accordance with the Journal of Laws No 48. The material for the research was blood plasma obtained after centrifuging full, nonhemolyzed blood. The blood was collected from the zygomatic vein directly to the test tubes with EDTA and transported in cooling conditions to the laboratory. After transporting the samples of blood to a certified analytical laboratory, the following elements of the obtained blood plasma were examined: ceruloplasmin . using turbidimetric method; transferrin . using immunoturbimetric method; troponin- using a third generation assay on an Elecsys; total protein, albumin, globulin . using spectrophotometric method and total iron . using colorimetric method. The results were statistically analyzed, i.e. the correlation between the parameters was measured by means of Pearsonfs correlation coefficient. The analysis of the results revealed a number of statistically significant relations between the parameters under the investigation, especially among the compounds directly responsible for metabolism of iron and copper. A statistically important positive correlation was observed between ceruloplasmin and ferritin (r = 0.563; P.0.05 and a negative one between transferrin and troponin (r = -0.609; P.0.05. Moreover, the content of transferrin . an iron-binding protein . was 0.17 g/l, while the concentration of iron was 58 ƒĘmol/l. The content of ceruloplasmin . a protein responsible for metabolism of copper . was very low (0.036 g/l. The level of proteins in the blood plasma of the animals under the research was approximately 72 g/l, with the share of albumins about 46%. The albumin-globulin ratio was 0.86.

  9. Blue native protein electrophoresis for studies of mouse polyomavirus morphogenesis and interactions between the major capsid protein VP1 and cellular proteins

    Czech Academy of Sciences Publication Activity Database

    Horníková, L.; Man, Petr; Forstová, J.

    2011-01-01

    Roč. 178, 1-2 (2011), s. 229-234 ISSN 0166-0934 R&D Projects: GA MŠk LC545 Institutional research plan: CEZ:AV0Z50200510 Keywords : BN-PAGE * Mouse polyomavirus * VP1 protein Subject RIV: CE - Biochemistry Impact factor: 2.011, year: 2011

  10. The action of red scorpion (Mesobuthus tamulus coconsis, pocock venom and its isolated protein fractions on blood sodium levels

    Directory of Open Access Journals (Sweden)

    R. V. Badhe

    2007-01-01

    Full Text Available Red scorpion (Mesobuthus tamulus or Buthus tamulus venom samples were collected at different regions of India: western (Chiplun and Ahmednagar from Maharashtra State and southern (Ratnagiri and Chennai from Tamil Nadu State. The action of whole venoms on the blood sodium levels of mice was assessed using flame photometry. Seven peptides were common to all venom samples. They were separated using the native polyacrylamide gel electrophoresis (PAGE technique and their activities were also studied using flame photometry. There was a decrease in the concentration of sodium ions in the serum, which suggested the blockage of such ions by scorpion venom toxins. Among the 10 protein bands isolated, the band at 79.6 kDa presented maximum activity in decreasing serum sodium ions concentration. Whole venom from Chiplun region also showed maximum activity. The western blotting technique demonstrated that the anti-scorpion venom sera produced by Haffkine Biopharmaceuticals Corporation Ltd., India, neutralized all four venom samples.

  11. Proteomic analysis of differential protein expression of achilles tendon in a rabbit model by two-dimensional polyacrylamide gel electrophoresis at 21 days postoperation.

    Science.gov (United States)

    Jielile, Jiasharete; Jialili, Ainuer; Sabirhazi, Gulnur; Shawutali, Nuerai; Redati, Darebai; Chen, Jiangtao; Tang, Bin; Bai, Jingping; Aldyarhan, Kayrat

    2011-10-01

    Postoperative early kinesitherapy has been advocated as an optimal method for treating Achilles tendon rupture. However, an insight into the rationale of how early kinesitherapy contributes to healing of Achilles tendon remains to be achieved, and research in the area of proteomic analysis of Achilles tendon has so far been lacking. Forty-two rabbits were randomized into control group, immobilization group, and early motion group, and received postoperative cast immobilization and early motion treatments. Achilles tendon samples were prepared 21 days following microsurgery, and the proteins were separated with two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were first recognized by PDQuest software, and then identified using peptide mass fingerprinting, tandem mass spectrometry, and database searching. A total of 463  ±  12, 511  ±  39, and 513  ±  80 protein spots were successfully detected in the two-dimensional polyacrylamide gels for the Achilles tendon samples of rabbits in the control group, immobilization group, and early motion group, respectively. There were 15, 8, and 9 unique proteins in these three groups, respectively, and some differentially expressed proteins were also identified in each group. It was indicated that some of the differentially expressed proteins were involved in various metabolism pathways and may play an important role in healing of Achilles tendon rupture. Postoperative early kinesitherapy resulted in differentially expressed proteins in ruptured Achilles tendon compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to healing of Achilles tendon rupture through a mechanobiological mechanism due to the application of postoperative early kinesitherapy.

  12. Novel blood protein based scaffolds for cardiovascular tissue engineering

    Directory of Open Access Journals (Sweden)

    Kuhn Antonia I.

    2016-09-01

    Full Text Available A major challenge in cardiovascular tissue engineering is the fabrication of scaffolds, which provide appropriate morphological and mechanical properties while avoiding undesirable immune reactions. In this study electrospinning was used to fabricate scaffolds out of blood proteins for cardiovascular tissue engineering. Lyophilised porcine plasma was dissolved in deionised water at a final concentration of 7.5% m/v and blended with 3.7% m/v PEO. Electrospinning resulted in homogeneous fibre morphologies with a mean fibre diameter of 151 nm, which could be adapted to create macroscopic shapes (mats, tubes. Cross-linking with glutaraldehyde vapour improved the long-term stability of protein based scaffolds in comparison to untreated scaffolds, resulting in a mass loss of 41% and 96% after 28 days of incubation in aqueous solution, respectively.

  13. Pharmaceutical protein production by yeast: towards production of human blood proteins by microbial fermentation

    DEFF Research Database (Denmark)

    Martinez Ruiz, José Luis; Liu, Lifang; Petranovic, Dina

    2012-01-01

    techniques (the so-called—omics approaches) and integrative approaches (systems biology) allow the development of novel microbial cell factories as valuable platforms for large scale production of therapeutic proteins. This review summarizes the main achievements and the current situation in the field...... of recombinant therapeutics using yeast Saccharomyces cerevisiae as a model platform, and discusses the future potential of this platform for production of blood proteins and substitutes.......Since the approval of recombinant insulin from Escherichia coli for its clinical use in the early 1980s, the amount of recombinant pharmaceutical proteins obtained by microbial fermentations has significantly increased. The recent advances in genomics together with high throughput analysis...

  14. Diagnostic implications of enumerating and reporting beta fraction(s) for the detection of beta-migrating monoclonal immunoglobulins in serum protein electrophoresis.

    Science.gov (United States)

    Chan, Pak Cheung; Lem-Ragosnig, Bonny; Chen, Jialun

    2018-03-01

    Modern serum protein electrophoresis (SPE) resolves serum proteins in 6 major fractions, splitting beta (B) into beta1 (B1) and beta2 (B2). Beta-migrating monoclonal immunoglobulins (B-MC) commonly integrate into normal-looking peak shape(s) but may increase the fraction value, forming the basis for reflex testing. The objectives of this study were (1) to ascertain the value of beta fraction(s) reporting, and (2) to compare the diagnostic performance between different beta-flagging approaches, particularly ↑B versus ↑B1 and/or ↑B2. We retrospectively studied 22,900 consecutive SPEs, and identified 3974 paired SPE and immunofixation electrophoresis (IFE) results obtained from the Sebia Capillarys™ 2 and Hydrasys™ electrophoresis systems respectively. ↑B, ↑B1 and ↑B2 were defined as fraction concentrations >11, 6 and 5 g/L respectively, and their respective diagnostic metrics calculated using IFE as the reference standard. 32 beta-gamma bridges were B-MC negative and thus excluded. Of 3942 cases, 142, 18, 285 and 300 had ↑B, ↑B1, ↑B2 and ↑B1 and/or ↑B2 respectively, while their corresponding sensitivities for B-MC were 0.38, 0.09, 0.45 and 0.54 respectively. Comparing ↑B and ↑B1 and/or ↑B2, ↑B had significantly lower sensitivity but higher specificity (0.98 Vs 0.95) and positive predictive value (0.47 Vs 0.31). All 4 beta fraction increases had odds ratios ranged from 14 to 118. Beta increases were associated with increased odds for B-MC, hence providing value and justification for their reporting and reflex testing. ↑B1 and/or ↑B2 detected more B-MC than ↑B, supporting separate reporting of B1 and B2. Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  15. Oral delivery of bioencapsulated proteins across blood-brain and blood-retinal barriers.

    Science.gov (United States)

    Kohli, Neha; Westerveld, Donevan R; Ayache, Alexandra C; Verma, Amrisha; Shil, Pollob; Prasad, Tuhina; Zhu, Ping; Chan, Sic L; Li, Qiuhong; Daniell, Henry

    2014-03-01

    Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood-brain barriers (BBB) and blood-retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S-stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid β 42 (Aβ42) to soluble fractions. CTB-MBP oral delivery reduced Aβ42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB.

  16. Misleading presentation of haemoglobin electrophoresis data | Adu ...

    African Journals Online (AJOL)

    Haemoglobinopathies are common in sub-Saharan Africa. As such haemoglobin electrophoresis are required to inform clinical decision making. However, haemoglobin electrophoresis is an assay that detects protein at either alkaline or acidic pH. Such assays do not interrogate gene sequences but rather the product of a ...

  17. misleading presentation of haemoglobin electrophoresis data

    African Journals Online (AJOL)

    SUMMARY. Haemoglobinopathies are common in sub-Saharan Africa. As such haemoglobin electrophoresis are required to in- form clinical decision making. However, haemoglobin electrophoresis is an assay that detects protein at either alka- line or acidic pH. Such assays do not interrogate gene sequences but rather ...

  18. Investigation on accordance of DNA double-strand break of blood between in vivo and in vitro irradiation using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Liu Qiang; Jiang Enhai; Li Jin; Tang Weisheng; Wang Zhiquan; Zhao Yongcheng; Fan Feiyue

    2006-01-01

    Objective: To observe the consistency of DNA double-strand break between in vivo and in vitro irradiation, as a prophase study in radiation biodosimetry using single cell gel electrophoresis (SCGE). Methods: Detect DNA double-strand break after whole-body and in vitro radiation in mice lymphocytes using neutral single cell gel electrophoresis. The comet images were processed by CASP software and all the data were analysed by SPSS12.0. Results: There is no difference between in vivo and in vitro irradiation group in HDNA%, TDNA%, CL, TL, TM and OTM. Conclusion: The result of neutral single cell gel electrophoresis shortly after in vitro irradiation can precisely reflect the DNA double-strand break of lymphocytes in whole-body irradiation. (authors)

  19. A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.

    Science.gov (United States)

    Makinde, A A; Gyles, C L

    1999-07-01

    Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences

  20. Early quantitative profiling of differential retinal protein expression in lens-induced myopia in guinea pig using fluorescence difference two-dimensional gel electrophoresis.

    Science.gov (United States)

    Wu, Yi; Lam, Carly Siu-Yin; Tse, Dennis Yan-Yin; To, Chi Ho; Liu, Quan; McFadden, Sally A; Chun, Rachel Ka-Man; Li, King Kit; Bian, Jianfang; Lam, Chuen

    2018-04-01

    The current study aimed to investigate the differential protein expression in guinea pig retinas in response to lens-induced myopia (LIM) before fully compensated eye growth. Four days old guinea pigs (n=5) were subjected to ‑4D LIM for 8 days. Refractive errors were measured before and at the end of the lens wear period. Ocular dimensions were also recorded using high‑frequency A‑scan ultrasonography. After the LIM treatment, retinas of both eyes were harvested and soluble proteins were extracted. Paired retinal protein expressions in each animal were profiled and compared using a sensitive fluorescence difference two‑dimensional gel electrophoresis. The quantitative retinal proteomes of myopic and control eye were analysed using computerised DeCyder software. Those proteins that were consistently changed with at least 1.2‑fold difference (P<0.05) in the same direction in all five animals were extracted, trypsin digested and identified by tandem mass spectrometry. Significant myopia was induced in guinea pigs after 8 days of lens wear. The vitreous chamber depth in lens‑treated eyes was found to be significantly elongated. Typically, more than 1,000 protein spots could be detected from each retina. Thirty‑two of them showed differential expression between myopic and untreated retina. Among these proteins, 21 spots were upregulated and 11 were downregulated. Eight protein spots could be successfully identified which included β‑actin, enolase 1, cytosolic malate dehydrogenase, Ras‑related protein Rab‑11B, protein‑L‑isoaspartate (D‑aspartate) O‑methyltransferase, PKM2 protein, X‑linked eukaryotic translation initiation factor 1A and ACP1 protein. The present study serves as the first report to uncover the retinal 2D proteome expressions in mammalian guinea pig myopia model using a top‑down fluorescent dyes labelling gel approach. The results showed a downregulation in glycolytic enzymes that may suggest a significant alteration of

  1. Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

    Science.gov (United States)

    Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

    2004-09-01

    We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

  2. A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

    Science.gov (United States)

    Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

    2011-07-01

    Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

  3. Primary Separation: 2-D Electrophoresis

    NARCIS (Netherlands)

    Pedreschi Plasencia, R.P.

    2013-01-01

    The advancements in two-dimensional electrophoresis (2DE) have consolidated it as a key tool for gel-based proteomics applications. Nowadays, 2DE is extensively applied and it is a useful technique for the simultaneous separation of hundreds to thousands of proteins, analysis, and differential

  4. HP-Lattice QSAR for dynein proteins: experimental proteomics (2D-electrophoresis, mass spectrometry) and theoretic study of a Leishmania infantum sequence.

    Science.gov (United States)

    Dea-Ayuela, María Auxiliadora; Pérez-Castillo, Yunierkis; Meneses-Marcel, Alfredo; Ubeira, Florencio M; Bolas-Fernández, Francisco; Chou, Kuo-Chen; González-Díaz, Humberto

    2008-08-15

    The toxicity and inefficacy of actual organic drugs against Leishmaniosis justify research projects to find new molecular targets in Leishmania species including Leishmania infantum (L. infantum) and Leishmaniamajor (L. major), both important pathogens. In this sense, quantitative structure-activity relationship (QSAR) methods, which are very useful in Bioorganic and Medicinal Chemistry to discover small-sized drugs, may help to identify not only new drugs but also new drug targets, if we apply them to proteins. Dyneins are important proteins of these parasites governing fundamental processes such as cilia and flagella motion, nuclear migration, organization of the mitotic splinde, and chromosome separation during mitosis. However, despite the interest for them as potential drug targets, so far there has been no report whatsoever on dyneins with QSAR techniques. To the best of our knowledge, we report here the first QSAR for dynein proteins. We used as input the Spectral Moments of a Markov matrix associated to the HP-Lattice Network of the protein sequence. The data contain 411 protein sequences of different species selected by ClustalX to develop a QSAR that correctly discriminates on average between 92.75% and 92.51% of dyneins and other proteins in four different train and cross-validation datasets. We also report a combined experimental and theoretic study of a new dynein sequence in order to illustrate the utility of the model to search for potential drug targets with a practical example. First, we carried out a 2D-electrophoresis analysis of L. infantum biological samples. Next, we excised from 2D-E gels one spot of interest belonging to an unknown protein or protein fragment in the region Mdata base with the highest similarity score to the MS of the protein isolated from L. infantum. We used the QSAR model to predict the new sequence as dynein with probability of 99.99% without relying upon alignment. In order to confirm the previous function annotation we

  5. The Cutting Edge of Affinity Electrophoresis Technology

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  6. The Cutting Edge of Affinity Electrophoresis Technology.

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-03-18

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  7. Evaluation and comparison of four protein extraction protocols for mono- and two-dimensional electrophoresis in Mytilus galloprovincialis

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2015-09-01

    Full Text Available In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i TRIzol reagent; ii Lysis buffer; iii phenylmethanesulfonyl fluoride; iv trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis.

  8. Characterization of cyst and trophozoite proteins of environmental isolates of Acanthamoeba castellanii by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Dao, Yap Ray Ming; Chooi, Lim Patricia Kim; Li, Chan Li; Fung, Wong Shew; Wah, Mak Joon

    2014-03-01

    Acanthamoeba castellanii has been known to possess pathogenic properties, such as acanthamoebic keratitis and granulomatous amoebic encephalitis. The role of proteases and proteins in the pathogenesis of these infections is still poorly understood. As Acanthamoeba sp is a ubiquitous protozoon found in the natural environment they can potentially cause human infections. This study characterized cyst and trophozoite proteins of 3 environmental A. castellanii isolates in comparison with a clinical isolate, ATCC 50492. The latter and environmental IMU1 isolate had 100% genotype identity with A. castellanii and demonstrated protein spots with higher molecular weights (> 95 kDa) at relatively higher isoelectric values (> pI 7.00) compared to the two other environmental isolates (IMU4 and IMU5) that had 99% genotype identity to A. castellanii based on 16 S rDNA sequence. Thus such trophozoite proteins may be involved with the parasite's ability to cause acanthamoebic keratitis.

  9. Supported liquid membrane extraction coupled in-line to commercial capillary electrophoresis for rapid determination of formate in undiluted blood samples

    Czech Academy of Sciences Publication Activity Database

    Pantůčková, Pavla; Kubáň, Pavel; Boček, Petr

    2013-01-01

    Roč. 1299, JUL (2013), s. 33-39 ISSN 0021-9673 R&D Projects: GA ČR(CZ) GA13-05762S Institutional support: RVO:68081715 Keywords : capillary electrophoresis * supported liquid membranes * methanol intoxication Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.258, year: 2013

  10. Direct analysis of formate in human plasma, serum and whole blood by in-line coupling of microdialysis to capillary electrophoresis for rapid diagnosis of methanol poisoning

    Czech Academy of Sciences Publication Activity Database

    Kubáň, Pavel; Boček, Petr

    2013-01-01

    Roč. 768, 21 JAN (2013), s. 82-89 ISSN 0003-2670 R&D Projects: GA ČR GAP206/10/1219 Institutional support: RVO:68081715 Keywords : capillary electrophoresis * in-line microdialysis * methanol intoxication Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.517, year: 2013

  11. Protein-associated water and secondary structure effect removal of blood proteins from metallic substrates.

    Science.gov (United States)

    Anand, Gaurav; Zhang, Fuming; Linhardt, Robert J; Belfort, Georges

    2011-03-01

    Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins

  12. In-Depth Characterization of the Phaseolin Protein Diversity of Common Bean (Phaseolus vulgaris L. Based on Two-Dimensional Electrophoresis and Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    María López-Pedrouso

    2012-01-01

    Full Text Available Phaseolin is the major seed storage protein of common bean. It comprises a complex set of glycoproteins heterogeneous in their polypeptide composition that is encoded by a gene family. Analyses of phaseolin banding patterns by one-dimensional electrophoresis (SDS-PAGE have been central to the current understanding of the diversity of wild and cultivated common beans. In this work, we have carried out a detailed description and interpretation of phaseolin diversity in cultivated common beans of different geographic origins (Mesoamerican and Andean gene pools based on the current two-dimensional electrophoresis (2-DE technology and mass spectrometry (MS. High-quality 2-DE gel images revealed very complex phaseolin patterns across the studied cultivars. Specifically, patterns of phaseolin within cultivars were organized in a horizontal string of multiple isospot pairs varying in isoelectric point and molecular mass. The degree of similarity among phaseolin patterns was estimated from the percentage of spots shared between pairs of cultivars. Analyses of proteomic distances between phaseolin types by non-metrical multidimensional scaling revealed that 2-DE phaseolin profiles are more similar among cultivars belonging to the same gene pool. However, higher differentiation was found among cultivars of the Andean gene pool. Analysis of genetic variations of the PCR-based SCAR marker of phaseolin seed protein was in general agreement with 2-DE phaseolin patterns, but provided supplementary information regarding diversity among cultivars. Furthermore, the molecular basis responsible for the complexity of 2-DE phaseolin patterns was investigated. Thus, identification of phaseolin spots from 2-DE gels by MALDI-TOF and MALDI-TOF/TOF MS showed that each single isospot pair contained only one type (α or β of phaseolin polypeptide, but pairs with higher and lower molecular mass corresponded to α- and β-type polypeptides, respectively. In addition, partial

  13. Evaluation of wheat by polyacrylamide gel electrophoresis

    African Journals Online (AJOL)

    PRECIOUS

    2010-01-11

    04, Inqulab-91 and Rawal-. 87 were evaluated for analysis of variability in seed storage proteins by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophorogram for each variety were scored ...

  14. Insoluble eggshell matrix proteins - their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatography

    Czech Academy of Sciences Publication Activity Database

    Mikšík, Ivan; Charvátová, Jana; Eckhardt, Adam; Deyl, Zdeněk

    2003-01-01

    Roč. 24, č. 5 (2003), s. 843-852 ISSN 0173-0835 R&D Projects: GA ČR GA203/02/1467; GA ČR GA203/00/D032 Institutional research plan: CEZ:AV0Z5011922 Keywords : eggshell matrix protein * peptide mapping Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.040, year: 2003

  15. In-line coupling of microextractions across polymer inclusion membranes to capillary zone electrophoresis for rapid determination of formate in blood samples

    Czech Academy of Sciences Publication Activity Database

    Pantůčková, Pavla; Kubáň, Pavel; Boček, Petr

    2015-01-01

    Roč. 887, AUG (2015), s. 111-117 ISSN 0003-2670 R&D Projects: GA ČR(CZ) GA13-05762S Grant - others:GA AV ČR(CZ) R200311404 Institutional support: RVO:68081715 Keywords : capillary electrophoresis * in- line coupling * polymer inclusion membrane extraction Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.712, year: 2015

  16. In-line coupling of microextractions across polymer inclusion membranes to capillary zone electrophoresis for rapid determination of formate in blood samples

    Czech Academy of Sciences Publication Activity Database

    Pantůčková, Pavla; Kubáň, Pavel; Boček, Petr

    2015-01-01

    Roč. 887, AUG (2015), s. 111-117 ISSN 0003-2670 R&D Projects: GA ČR(CZ) GA13-05762S Grant - others:GA AV ČR(CZ) R200311404 Institutional support: RVO:68081715 Keywords : capillary electrophoresis * in-line coupling * polymer inclusion membrane extraction Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.712, year: 2015

  17. Phylogenetic relationship among five geckos from Egypt based on RAPD-PCR and protein electrophoresis (SDS–PAGE

    Directory of Open Access Journals (Sweden)

    Nadia H.M. Sayed

    2012-05-01

    Full Text Available Genetic variations between five gekkonid species from Egypt; Tropiocolotes tripolitanus, Tropiocolotes steudneri, Tropiocolotes nattereri, Tarentola mauritanica and Tarentola annularis were analyzed by SDS–PAGE for water soluble proteins and random amplified polymorphic DNA (RAPD analysis. Based on SDS–PAGE of water soluble proteins for all species, the obtained results revealed a total of 17 bands at molecular weights that ranged from 95 to 16 kDa. The polymorphic bands among species were 11 (64.7% and the mean similarity matrix value between them was 70.7%. Using RAPD-PCR, the results showed eight total amplified bands at molecular weights that ranged from 1408 to 360 bp. The polymorphic bands between species were 7 (87.5% and the mean similarity matrix between them was 44.6%. The dendrogram showed that, the five gekkonid species are separated from each other into two clusters. The first cluster contains three species of the genus Tropiocolotes. The second cluster includes the two species of the genus Tarentola. Based on SDS–PAGE and RAPD-PCR results, T. nattereri is sister to T. steudneri with higher genetic similarity than with T. tripolitanus. It is concluded that, the similarity coefficient and the genetic distance values between the five gekkonid species indicate that the five gekkonid species are not identical and are separated from each other. From these results, it is indicated that the protein and RAPD analysis are useful molecular tools to indicate genetic variation between the species in the same genus or in the different genera.

  18. Phylogenetic analyses and detection of viridans streptococci based on sequences and denaturing gradient gel electrophoresis of the rod shape-determining protein gene

    Science.gov (United States)

    Konishi, Ikuri; Hoshino, Tomonori; Kondo, Yoshio; Saito, Kan; Nishiguchi, Miyuki; Sato, Kyoko; Fujiwara, Taku

    2009-01-01

    Background Population analysis of viridans streptococci is important because these species are associated with dental caries, bacteremia, and subacute endocarditis, in addition to being important members of the human oral commensal microbiota. Design In this study, we phylogenetically analyzed the rod shape-determining protein gene (rodA), which is associated with cellular morphology, cell division, and sensitivity for antibiotics, and demonstrated that the diversity of the rodA gene is sufficient to identify viridans streptococci at the species level. Moreover, we developed a more convenient denaturing gradient gel electrophoresis (DGGE) method based on the diversity of the rodA gene (rodA-DGGE) for detecting nine dominant streptococcal species in human saliva, namely, Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis, Streptococcus parasanguinis, Streptococcus gordonii, Streptococcus vestibularis, Streptococcus salivarius, Streptococcus mutans, and Streptococcus sobrinus. Results This rodA-DGGE method proved useful in detecting viridans streptococci without cultivation, isolation, and phenotypic characterization. Conclusion Analysis of the oral microbiota by rodA-DGGE offers a higher resolution than the conventional DGGE using 16S rDNA and may be an alternative in the microbial diagnosis of streptococcal infection. PMID:21523207

  19. Phylogenetic analyses and detection of viridans streptococci based on sequences and denaturing gradient gel electrophoresis of the rod shape-determining protein gene.

    Science.gov (United States)

    Konishi, Ikuri; Hoshino, Tomonori; Kondo, Yoshio; Saito, Kan; Nishiguchi, Miyuki; Sato, Kyoko; Fujiwara, Taku

    2009-08-28

    Population analysis of viridans streptococci is important because these species are associated with dental caries, bacteremia, and subacute endocarditis, in addition to being important members of the human oral commensal microbiota. In this study, we phylogenetically analyzed the rod shape-determining protein gene (rodA), which is associated with cellular morphology, cell division, and sensitivity for antibiotics, and demonstrated that the diversity of the rodA gene is sufficient to identify viridans streptococci at the species level. Moreover, we developed a more convenient denaturing gradient gel electrophoresis (DGGE) method based on the diversity of the rodA gene (rodA-DGGE) for detecting nine dominant streptococcal species in human saliva, namely, Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis, Streptococcus parasanguinis, Streptococcus gordonii, Streptococcus vestibularis, Streptococcus salivarius, Streptococcus mutans, and Streptococcus sobrinus. This rodA-DGGE method proved useful in detecting viridans streptococci without cultivation, isolation, and phenotypic characterization. Analysis of the oral microbiota by rodA-DGGE offers a higher resolution than the conventional DGGE using 16S rDNA and may be an alternative in the microbial diagnosis of streptococcal infection.

  20. Assessment of genetic diversity among some accessions of sage (Salvia officinalis L. using electrophoresis of seed storage proteins

    Directory of Open Access Journals (Sweden)

    Seyed Abbas Mirjalili

    2016-06-01

    Full Text Available The genus Salvia (Lamiaceae comprises over 900 species in the world, with a relatively wide dispersion in the Iran’s flora. Until now, about 58 species of the genus have been reported and identified in Iran, in which 17 of them were endemic. In order to study, investigate and evaluate the intraspecific diversity, similarity and dissimilarity among Iranian Salvia officinalis accessions, an experiment was carried out using SDS-PAGE technique. In this study, the seeds from five accessions were collected from gene bank and were evaluated. The seed storage proteins were extracted by buffers and were measured. Phylogenetic relationships were analyzed according to presence and absence of bands on the gel. A dendrogram was prepared using calculation of the accession’s similarity index. Mean comparison were done by Tukey’s test. The seed protein contents showed significant differences (p≤0.01 among accessions. A total of 39 bands were indicated on the gel. The maximum diversity was detected in the accession No. 2 while, the lowest band’s number were recorded with the accessions No. 1 and 5. Based on dendrogram, the accessions were divided into two groups; one includes accession No. 1 and 4 other accessions were located in the second group further classified into two subgroup including accessions No. 2 and 3 in one clade and accessions No. 4 and 5 in the other ones.

  1. Perfil eletroforético das proteínas séricas de serpentes Crotalus durissus terrificus (cascavel criadas em cativeiro Serum protein electrophoresis profile of the rattlesnake Crotalus durissus terrificus kept in captivity

    Directory of Open Access Journals (Sweden)

    Joandes Henrique Fonteque

    2009-06-01

    Full Text Available As serpentes peçonhentas dos gêneros Bothrops e Crotalus têm sido mantidas em cativeiro visando à extração de venenos para a produção de imunobiológicos. O conhecimento da fisiologia desses animais e as alterações na concentração de proteínas e suas frações séricas são importantes para a identificação precoce de importantes enfermidades que cursam com estados de hipoproteinemia e hiperproteinemia. O objetivo do trabalho foi determinar a concentração de proteína total e o perfil eletroforético das proteínas séricas de serpentes Crotalus durissus terrificus (cascavel criadas em cativeiro. Foram colhidas amostras de sangue da veia coccígea ventral de 21 serpentes adultas e sadias, divididas em dois grupos: Grupo 1 de 12 machos com peso médio de 588,89±193,55g, e Grupo 2 de nove fêmeas com peso médio de 708,33±194,04g. A proteína total sérica foi determinada pelo método de refratometria e a eletroforese em gel de agarose. Obtiveram-se valores da proteína total sérica (g/dL de 4,51±0,50 para machos e de 4,82±0,72 para fêmeas, e para machos e fêmeas de 4,64±0,61. Foram identificadas pela eletroforese quatro frações protéicas (g/dL: albumina, a, b, g-globulinas e calculada a relação albumina:globulina. As serpentes fêmeas apresentaram maiores valores para as variáveis, albumina e para a relação albumina/globulina (AG diferindo significativamente (PThe poisonous snakes of the genera Crotalus and Bothrops have been kept in captivity with the purpose of extracting poison for the production of immunobiological. Knowledge of the physiology of these animals and serum proteins concentration changes are important for early identification of major diseases which lead to states of hypoproteinemia and hyperproteinemia. The objective was to determine the concentration of total protein and serum protein electrophoresis profile of Crotalus durissus terrificus (rattlesnake in captivity. Blood samples were taken from

  2. Research on pre-staining gel electrophoresis

    International Nuclear Information System (INIS)

    Zhong Ruibo; Liu Yushuang; Zhang Ping; Liu Jingran; Zhao Guofen; Zhang Feng

    2014-01-01

    Background: Gel electrophoresis is a powerful biochemical separation technique. Most biological molecules are completely transparent in the visible region of light, so it is necessary to use staining to show the results after gel electrophoresis, and the general steps of conventional staining methods are time-consuming. Purpose: We try to develop a novel approach to simplify the gel electrophoresis: Pre-Staining Gel Electrophoresis (PSGE), which can make the gel electrophoresis results monitored in real time. Methods: Pre-stain the protein samples with Coomassie Brilliant Blue (CBB) for 30 min before loading the sample into the gel well. Results and Conclusion: PSGE can be successfully used to analyze the binding efficiency of Bovine Serum Albumin (BSA) and amphiphilic polymer via chemical coupling and physical absorption, and the double PSGE also shows a great potential in bio-analytical chemistry. (authors)

  3. Blood Protein Polymorphism of Small Cattle Bred in Armenia

    Directory of Open Access Journals (Sweden)

    Gayane Marmaryan

    2017-05-01

    Full Text Available ABSTRACT Biochemical and genetic markers have not yet been used in selection and breeding of agricultural animals in Armenia. The objective behind the experiments was to assess the small cattle bred in Armenia- the semi-coarse wool and semi-fine wool sheep, as well as goats of different genotypes characterized by different milk productivity, polymorphic blood proteins-transferrin, ceruloplasmin, aiming to use them in breeding. Another objective was to study the genetic distance between the researched breeds and crossbreeds aiming to reveal the genetic similarity. The findings of research studies come to show that goats with polymorphic transferrin locus and a big set of genotypes are characterized by higher milk productivity; therefore it is recommended that they be used in selection as a supplementary milk productivity marker. The genetic distance between the researched goats comprised 0,60 which comes to show that semi-fine wool sheep were also bred with the help of semi-coarse wool crossbreeds. The high coefficients of genetic distance of crossbreed goats compared to both the highly milk producing imported breeds and the locals are indicative of inherited high adaptability qualities of locals and high milk producing quality of pure breeds.

  4. Levels of Blood Glucose and Total Protein of Repeat Breeding Dairy Cows From Daerah Istimewa Yogyakarta

    Directory of Open Access Journals (Sweden)

    Dhasia Ramandani

    2015-11-01

    breeding case had lower blood glucose and total protein plasm concentrations than that of the normal. The average concentrations of blood glucose and total protein plasm were 48.58±6.675 mg/dl and 6.815±821 g/dl, respectively.

  5. Polyacrylamide temperature gradient gel electrophoresis.

    Science.gov (United States)

    Viglasky, Viktor

    2013-01-01

    Temperature Gradient Gel Electrophoresis (TGGE) is a form of electrophoresis in which temperature gradient is used to denature molecules as they move through either acrylamide or agarose gel. TGGE can be applied to analyze DNA, RNA, protein-DNA complexes, and, less commonly, proteins. Separation of double-stranded DNA molecules during TGGE relies on temperature-dependent melting of the DNA duplex into two single-stranded DNA molecules. Therefore, the mobility of DNA reflects not only the size of the molecule but also its nucleotide composition, thereby allowing separation of DNA molecules of similar size with different sequences. Depending on the relative orientation of electric field and temperature gradient, TGGE can be performed in either a parallel or a perpendicular mode. The former is used to analyze multiple samples in the same gel, whereas the later allows detailed analysis of a single sample. This chapter is focused on analysis of DNA by polyacrylamide TGGE using the perpendicular mode.

  6. Surfactant Protein D Levels in Umbilical Cord Blood and Capillary Blood of Premature Infants

    DEFF Research Database (Denmark)

    Dahl, Marianne; Holmskov, Uffe; Husby, Steffen

    2006-01-01

    and capillary blood was measured using ELISA technique. The median concentration of SP-D in umbilical cord blood was twice as high as in mature infants, 769 ng/mL (range 140-2,551), with lowest values in infants with intrauterine growth retardation (IUGR) and rupture of membranes (ROM). The median concentration...... of premature infants and to relate the levels to perinatal conditions. A total of 254 premature infants were enrolled in the present study. Umbilical cord blood was drawn at the time of birth and capillary blood at regular intervals throughout the admission. The concentration of SP-D in umbilical cord blood...... treatment and susceptibility to infections. We conclude that SP-D concentrations in umbilical cord blood and capillary blood in premature infants are twice as high as in mature infants and depend on several perinatal conditions. High SP-D levels in umbilical cord blood and capillary blood on day 1 were...

  7. Relative Abundance of Proteins in Blood Plasma Samples from Patients with Chronic Cerebral Ischemia.

    Science.gov (United States)

    Kaysheva, Anna L; Kopylov, Artur T; Ponomarenko, Elena A; Kiseleva, Olga I; Teryaeva, Nadezhda B; Potapov, Alexander A; Izotov, Alexander А; Morozov, Sergei G; Kudryavtseva, Valeria Yu; Archakov, Alexander I

    2018-03-01

    A comparative protein profile analysis of 17 blood plasma samples from patients with ischemia and 20 samples from healthy volunteers was carried out using ultra-high resolution mass spectrometry. The analysis of measurements was performed using the proteomics search engine OMSSA. Normalized spectrum abundance factor (NSAF) in the biological samples was assessed using SearchGUI. The findings of mass spectrometry analysis of the protein composition of blood plasma samples demonstrate that the depleted samples are quite similar in protein composition and relative abundance of proteins. By comparing them with the control samples, we have found a small group of 44 proteins characteristic of the blood plasma samples from patients with chronic cerebral ischemia. These proteins contribute to the processes of homeostasis maintenance, including innate immune response unfolding, the response of a body to stress, and contribution to the blood clotting cascade.

  8. Effects of protein intake on blood pressure, insulin sensitivity and blood lipids in children: a systematic review.

    Science.gov (United States)

    Voortman, Trudy; Vitezova, Anna; Bramer, Wichor M; Ars, Charlotte L; Bautista, Paula K; Buitrago-Lopez, Adriana; Felix, Janine F; Leermakers, Elisabeth T M; Sajjad, Ayesha; Sedaghat, Sanaz; Tharner, Anne; Franco, Oscar H; van den Hooven, Edith H

    2015-02-14

    High protein intake in early childhood is associated with obesity, suggesting possible adverse effects on other cardiometabolic outcomes. However, studies in adults have suggested beneficial effects of protein intake on blood pressure (BP) and lipid profile. Whether dietary protein intake is associated with cardiovascular and metabolic health in children is unclear. Therefore, we aimed to systematically review the evidence on the associations of protein intake with BP, insulin sensitivity and blood lipids in children. We searched the databases Medline, Embase, Cochrane Central and PubMed for interventional and observational studies in healthy children up to the age of 18 years, in which associations of total, animal and/or vegetable protein intake with one or more of the following outcomes were reported: BP; measures of insulin sensitivity; cholesterol levels; or TAG levels. In the search, we identified 6636 abstracts, of which fifty-six studies met all selection criteria. In general, the quality of the included studies was low. Most studies were cross-sectional, and many did not control for potential confounders. No overall associations were observed between protein intake and insulin sensitivity or blood lipids. A few studies suggested an inverse association between dietary protein intake and BP, but evidence was inconclusive. Only four studies examined the effects of vegetable or animal protein intake, but with inconsistent results. In conclusion, the literature, to date provides insufficient evidence for effects of protein intake on BP, insulin sensitivity or blood lipids in children. Future studies could be improved by adequately adjusting for key confounders such as energy intake and obesity.

  9. Electrophoretic variants of blood proteins in Japanese, 7

    International Nuclear Information System (INIS)

    Satoh, Chiyoko; Takahashi, Norio; Kimura, Yasukazu; Miura, Akiko; Kaneko, Junko; Fujita, Mikio; Toyama, Kyoko.

    1986-11-01

    A total of 16,835 children, of whom 11,737 are unrelated, from Hiroshima and Nagasaki were examined for erythrocyte cytoplasmic glutamate-oxaloacetate transaminase (GOT1) by starch gel electrophoresis. A variant allele named GOT1*2HR1 which seems to be identical with GOT1*2 was encountered in polymorphic frequency. Five kinds of rare variants, 3NG1, 4NG1, 5NG1, 6HR1, and 7NG1 were encountered in a total of 109 children. Except for 7NG1 for which complete family study was unable, family studies confirmed the genetic nature of these rare variants, since for all instances in which both parents could be examined, one of the parents exhibited the same variant as that of their child. Thermostability profiles of these six variants were normal. The enzyme activities of five were decreased, while the value of one was normal compared to that of GOT1 1. (author)

  10. Differential effects of proteins and carbohydrates on postprandial blood pressure-related responses

    NARCIS (Netherlands)

    Teunissen-Beekman, Karianna F. M.; Dopheide, Janneke; Geleijnse, Johanna M.; Bakker, Stephan J. L.; Brink, Elizabeth J.; de Leeuw, Peter W.; Serroyen, Jan; van Baak, Marleen A.

    2014-01-01

    Diet composition may affect blood pressure (BP), but the mechanisms are unclear. The aim of the present study was to compare postprandial BP-related responses to the ingestion of pea protein, milk protein and egg-white protein. In addition, postprandial BP-related responses to the ingestion of

  11. The in vitro antioxidant properties of alcalase hydrolysate prepared from silkie fowl (Gallus gallus) blood protein.

    Science.gov (United States)

    Cheng, Fu-Yuan; Lai, I-Chun; Lin, Liang-Chuan; Sakata, Ryoichi

    2016-07-01

    Two types of proteins including blood plasma protein and blood cell protein were isolated from silkie fowl (Gallus gallus) blood and hydrolyzed using alcalase for 0, 2, 4 and 6 h. The blood plasma protein hydrolysate (BPH) and blood cell protein hydrolysate (BCH) were analyzed for pH value, peptide content and antioxidative properties. The significantly higher peptide contents were observed in BPH than that of BCH, which showed that blood plasma protein was more suitable to hydrolysis by alcalase than blood cell protein. Both BPH and BCH showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and Fe(2+) chelating ability. BPH at 4 h of hydrolysis (BPH4) demonstrated significantly higher antioxidant capacity than those treated by alcalase in most of the assays. The BPH4 was separated using ultra-filtration and assessment of the fractions and indicated that low molecular weight of peptides (< 3 kDa) possessed greater DPPH scavenging activity, Fe(2+) chelating ability and inhibitory activity of lipid peroxidation. These results show that BPH has the potential to be ingredients in the food industry as a replacement of synthetic antioxidants. © 2015 Japanese Society of Animal Science.

  12. Blood profiling of proteins and steroids during weight maintenance with manipulation of dietary protein level and glycaemic index

    DEFF Research Database (Denmark)

    Wang, Ping; Holst, Claus; Astrup, Arne

    2012-01-01

    Weight regain after weight loss is common. In the Diogenes dietary intervention study, a high-protein and low-glycaemic index (GI) diet improved weight maintenance. The objective of the present study was to identify (1) blood profiles associated with continued weight loss and weight regain (2......), evenly selected from four dietary groups that varied in protein and GI levels. The blood concentrations of twenty-nine proteins and three steroid hormones were measured. The changes in analytes during weight maintenance largely correlated negatively with the changes during weight loss, with some...

  13. Intake of total protein, plant protein and animal protein in relation to blood pressure : a meta-analysis of observational and intervention studies

    NARCIS (Netherlands)

    Tielemans, S. M. A. J.; Altorf-van der Kuil, W.; Engberink, M. F.; Brink, E. J.; van Baak, M. A.; Bakker, S. J. L.; Geleijnse, J. M.

    There is growing evidence from epidemiological studies that dietary protein may beneficially influence blood pressure (BP), but findings are inconclusive. We performed a meta-analysis of 29 observational studies and randomized controlled trials (RCTs) of dietary protein and types of protein in

  14. Disc electrophoresis and related techniques of polyacrylamide gel electrophoresis

    National Research Council Canada - National Science Library

    Maurer, H. R

    1971-01-01

    ..., enzymes, antingens and radioactively labelled materials, and detailed treatments of micro disc electrophoresis, preparative polyacrylamide gel electrophoresis and many other techniques for special problems...

  15. Concentration of Proteins and Protein Fractions in Blood Plasma of Chickens Hatched from Eggs Irradiated with Low Level Gamma Rays

    International Nuclear Information System (INIS)

    Kraljevic, P.; Vilic, M.; Simpraga, M.; Matisic, D.; Miljanic, S.

    2011-01-01

    In literature there are many results which have shown that low dose radiation can stimulate many physiological processes of living organism. In our earlier paper it was shown that low dose of gamma radiation has a stimulative effect upon metabolic process in chickens hatched from eggs irradiated before incubation. This was proved by increase of body weight gain and body weight, as well as by increase of two enzymes activities in blood plasma (aspartate aminotransferase and alanine aminotransferase) which play an important role in protein metabolism. Therefore, an attempt was made to determine the effect of eggs irradiation by low dose gamma rays upon concentration of total proteins and protein fractions in the blood plasma of chickens hatched from irradiated eggs. The eggs of heavy breed chickens were irradiated with a dose of 0.15 Gy gamma radiation (60Co) before incubation. Along with the chickens which were hatched from irradiated eggs, there was a control group of chickens hatched from nonirradiated eggs. All other conditions were the same for both groups of chickens. Blood samples were taken from the right jugular vein on the 1 s t and 3 r d day, or from the wing vein on days 5 and 7 after hatching. The total proteins concentration in the blood plasma was determined by the biuret method using Boehringer Mannheim GmbH optimized kits. The protein fractions (albumin, α 1 -globulin, α 2 -globulin, β- and γ-globulins) were estimated electrophoretically on Cellogel strips. The total proteins concentration was significantly decreased in blood plasma of chickens hatched from irradiated eggs on days 3 (P t h day (P 2 -globulin was decreased on days 1 (P t h day of life. Obtained results indicate that low dose of gamma radiation has mostly inhibitory effect upon concentration of total proteins and protein fractions in the blood plasma of chickens hatched from irradiated eggs before incubation. (author)

  16. Western blotting using capillary electrophoresis.

    Science.gov (United States)

    Anderson, Gwendolyn J; M Cipolla, Cynthia; Kennedy, Robert T

    2011-02-15

    A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.

  17. Audit of Use and Overuse of Serum Protein Immunofixation Electrophoresis and Serum Free Light Chain Assay in Tertiary Health Care: A Case for Algorithmic Testing to Optimize Laboratory Utilization.

    Science.gov (United States)

    Heaton, Christopher; Vyas, Shikhar G; Singh, Gurmukh

    2016-04-01

    Overuse of laboratory tests is a persistent issue. We examined the use and overuse of serum immunofixation electrophoresis and serum free light chain assays to develop an algorithm for optimizing utilization. A retrospective review of all tests, for investigation of monoclonal gammopathies, for all patients who had any of these tests done from April 24, 2014, through July 25, 2014, was carried out. The test orders were categorized as warranted or not warranted according to criteria presented in the article. A total of 237 patients were tested, and their historical records included 1,503 episodes of testing for one or more of serum protein electrophoresis, serum immunofixation electrophoresis, and serum free light chain assays. Only 46% of the serum immunofixation and 42% serum free light chain assays were warranted. Proper utilization, at our institution alone, would have obviated $64,182.95/year in health care costs, reduced laboratory cost of reagent alone by $26,436.04/year, and put $21,904.92/year of part B reimbursement at risk. Fewer than half of the serum immunofixation and serum free light chain assays added value. The proposed algorithm for testing should improve utilization. Risk to part B billing may be a disincentive to reducing test utilization. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent imp...... these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web....... the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make...

  19. Sources of dietary protein in relation to blood pressure in a general Dutch population

    NARCIS (Netherlands)

    Altorf-van der Kuil, W.; Engberink, M.F.; Vedder, M.M.; Boer, J.M.A.; Verschuren, W.M.M.; Geleijnse, J.M.

    2012-01-01

    Background - Little is known about the relation of different dietary protein types with blood pressure (BP). We examined whether intake of total, plant, animal, dairy, meat, and grain protein was related to BP in a cross sectional cohort of 20,820 Dutch adults, aged 20–65 y and not using

  20. Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines

    NARCIS (Netherlands)

    Krijgsveld, J.; Zaat, S. A.; Meeldijk, J.; van Veelen, P. A.; Fang, G.; Poolman, B.; Brandt, E.; Ehlert, J. E.; Kuijpers, A. J.; Engbers, G. H.; Feijen, J.; Dankert, J.

    2000-01-01

    Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their alpha-granules that are released upon thrombin activation. The present study was designed to

  1. Computational prediction of human salivary proteins from blood circulation and application to diagnostic biomarker identification.

    Directory of Open Access Journals (Sweden)

    Jiaxin Wang

    Full Text Available Proteins can move from blood circulation into salivary glands through active transportation, passive diffusion or ultrafiltration, some of which are then released into saliva and hence can potentially serve as biomarkers for diseases if accurately identified. We present a novel computational method for predicting salivary proteins that come from circulation. The basis for the prediction is a set of physiochemical and sequence features we found to be discerning between human proteins known to be movable from circulation to saliva and proteins deemed to be not in saliva. A classifier was trained based on these features using a support-vector machine to predict protein secretion into saliva. The classifier achieved 88.56% average recall and 90.76% average precision in 10-fold cross-validation on the training data, indicating that the selected features are informative. Considering the possibility that our negative training data may not be highly reliable (i.e., proteins predicted to be not in saliva, we have also trained a ranking method, aiming to rank the known salivary proteins from circulation as the highest among the proteins in the general background, based on the same features. This prediction capability can be used to predict potential biomarker proteins for specific human diseases when coupled with the information of differentially expressed proteins in diseased versus healthy control tissues and a prediction capability for blood-secretory proteins. Using such integrated information, we predicted 31 candidate biomarker proteins in saliva for breast cancer.

  2. A high confidence, manually validated human blood plasma protein reference set

    DEFF Research Database (Denmark)

    Schenk, Susann; Schoenhals, Gary J; de Souza, Gustavo

    2008-01-01

    , HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize......BACKGROUND: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list...... the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins. METHODS: To address the complexity and dynamic protein concentration range of the plasma proteome, we employed a linear ion...

  3. Multidimensional protein fractionation of blood proteins coupled to data-independent nanoLC-MS/MS analysis.

    Science.gov (United States)

    Levin, Yishai; Jaros, Julian A J; Schwarz, Emanuel; Bahn, Sabine

    2010-01-03

    In order to exploit human blood as a source of protein disease biomarkers, robust analytical methods are needed to overcome the inherent molecular complexity of this bio-fluid. We present the coupling of label-free SAX chromatography and IMAC to a data-independent nanoLC-MS/MS (nanoLC-MS(E)) platform for analysis of blood plasma and serum proteins. The methods were evaluated using protein standards added at different concentrations to two groups of samples. The results demonstrate that both techniques enable accurate protein quantitation using low sample volumes and a minimal number of fractions. Combining both methods, 883 unique proteins were identified, of which 423 proteins showed high reproducibility. The two approaches resulted in identification of unique molecular signatures with an overlap of approximately 30%, thus providing complimentary information on sub-proteomes. These methods are potentially useful for systems biology, biomarker discovery, and investigation of phosphoproteins in blood. (c) 2009 Elsevier B.V. All rights reserved.

  4. Gel Electrophoresis on a Budget to Dye for

    Science.gov (United States)

    Yu, Julie H.

    2010-01-01

    Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to $800 for each setup, which is cost prohibitive for most classroom budgets. This article…

  5. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Gouri Shankar Pandey

    2013-01-01

    Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

  6. Identification and comparative proteomic study of quail and duck egg white protein using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis.

    Science.gov (United States)

    Hu, S; Qiu, N; Liu, Y; Zhao, H; Gao, D; Song, R; Ma, M

    2016-05-01

    A proteomic study of egg white proteins from 2 major poultry species, namely quail (Coturnix coturnix) and duck (Anas platyrhynchos), was performed with comparison to those of chicken (Gallus gallus) through 2-dimensional polyacrylamide gel electrophoresis (2-DE) analysis. By using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), 29 protein spots representing 10 different kinds of proteins as well as 17 protein spots designating 9 proteins were successfully identified in quail and duck egg white, respectively. This report suggested a closer relationship between quail and chicken egg white proteome patterns, whereas the duck egg white protein distribution on the 2-DE map was more distinct. In duck egg white, some well-known major proteins, such as ovomucoid, clusterin, extracellular fatty acid-binding protein precursor (ex-FABP), and prostaglandin D2 synthase (PG D2 synthase), were not detected, while two major protein spots identified as "deleted in malignant brain tumors 1" protein (DMBT1) and vitellogenin-2 were found specific to duck in the corresponding range on the 2-DE gel map. These interspecies diversities may be associated with the egg white protein functions in cell defense or regulating/supporting the embryonic development to adapt to the inhabiting environment or reproduction demand during long-term evolution. The findings of this work will give insight into the advantages involved in the application on egg white proteins from various egg sources, which may present novel beneficial properties in the food industry or related to human health. © 2016 Poultry Science Association Inc.

  7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS ...

    African Journals Online (AJOL)

    Four strains of eri, Samia cynthia ricini Lepidoptera: Saturniidae that can be identified morphologically and maintained at North East Institute of Science and Technology, Jorhat were characterized based on their protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and DNA by random ...

  8. The relative nutritive value of irradiated spray-dried blood powder and heat-sterilized blood meal as measured in combination with whey protein

    International Nuclear Information System (INIS)

    Downes, T.E.H.; Nourse, L.D.; Siebrits, F.K.; Hastings, J.W.

    1987-01-01

    A method of processing blood meal in which nutritive value of the protein is preserved is described, since appreciable losses occur in the nutritive value of the protein when prepared by heat sterilization with drying at atmospheric pressure in steam jacketed vessels. Blood was spray dried and irradiated at an intensity of 10 kGy. Collectively the heat of spray drying and irradiation was effective in killing both the virus plaque-forming units and the bacteria, thus producing a commercially acceptable sterile product of higher nutritive value. The relative nutritive values (RNV) of 50:50 protein were 0,56 for whey protein concentrate plus heat-sterilized blood meal and 0.90 for whey protein concentrate plus irradiated spray-dried blood powder. Whey protein concentrate used as a control has a RNV of 1,0

  9. Lectin affinity electrophoresis.

    Science.gov (United States)

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  10. Blood-based protein biomarkers for diagnosis of Alzheimer disease.

    Science.gov (United States)

    Doecke, James D; Laws, Simon M; Faux, Noel G; Wilson, William; Burnham, Samantha C; Lam, Chiou-Peng; Mondal, Alinda; Bedo, Justin; Bush, Ashley I; Brown, Belinda; De Ruyck, Karl; Ellis, Kathryn A; Fowler, Christopher; Gupta, Veer B; Head, Richard; Macaulay, S Lance; Pertile, Kelly; Rowe, Christopher C; Rembach, Alan; Rodrigues, Mark; Rumble, Rebecca; Szoeke, Cassandra; Taddei, Kevin; Taddei, Tania; Trounson, Brett; Ames, David; Masters, Colin L; Martins, Ralph N

    2012-10-01

    To identify plasma biomarkers for the diagnosis of Alzheimer disease (AD). Baseline plasma screening of 151 multiplexed analytes combined with targeted biomarker and clinical pathology data. General community-based, prospective, longitudinal study of aging. A total of 754 healthy individuals serving as controls and 207 participants with AD from the Australian Imaging Biomarker and Lifestyle study (AIBL) cohort with identified biomarkers that were validated in 58 healthy controls and 112 individuals with AD from the Alzheimer Disease Neuroimaging Initiative (ADNI) cohort. A biomarker panel was identified that included markers significantly increased (cortisol, pancreatic polypeptide, insulinlike growth factor binding protein 2, β(2) microglobulin, vascular cell adhesion molecule 1, carcinoembryonic antigen, matrix metalloprotein 2, CD40, macrophage inflammatory protein 1α, superoxide dismutase, and homocysteine) and decreased (apolipoprotein E, epidermal growth factor receptor, hemoglobin, calcium, zinc, interleukin 17, and albumin) in AD. Cross-validated accuracy measures from the AIBL cohort reached a mean (SD) of 85% (3.0%) for sensitivity and specificity and 93% (3.0) for the area under the receiver operating characteristic curve. A second validation using the ADNI cohort attained accuracy measures of 80% (3.0%) for sensitivity and specificity and 85% (3.0) for area under the receiver operating characteristic curve. This study identified a panel of plasma biomarkers that distinguish individuals with AD from cognitively healthy control subjects with high sensitivity and specificity. Cross-validation within the AIBL cohort and further validation within the ADNI cohort provides strong evidence that the identified biomarkers are important for AD diagnosis.

  11. Capillary electrophoresis of diuretics.

    Science.gov (United States)

    Riekkola, M L; Jumppanen, J H

    1996-05-31

    The review surveys the application of capillary electrophoresis to the screening, identification and determination of diuretics and probenecid. The number of publications is still limited, but the studies already published clearly show that capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography are excellent alternatives for the investigation of diuretics. High accuracy identifications of diuretics and probenecid, even in urine samples, can be obtained when CZE is used with the marker techniques. This review paper has been written from the viewpoint of practical use and some hints are given for future CE studies on diuretics.

  12. Electrophoretic pattern of blood serum proteins of some of the vertebrates of Pakistan

    International Nuclear Information System (INIS)

    Shakoori, Abdul Rauf; Zaheer, Saleem Akhtar; Ahmad, Muhammad Salih.

    1976-01-01

    The electrophoretic pattern of blood serum proteins of some of the common fishes e.g. Catla catla, Cirrhina mrigala, Channa punctatus, Channa marulius, Wallago attu, Heterop-neustes fossilis; amphibia e.g., Rana tigrina, Rana cyanophlyctis, Bufo melanostictus; reptiles e.g. Varanus bengalensis, Uromastix hardwickii; birds e.g. Columba livia, Gallus domesticus, Passer domestica, Anas platyrhynchos; and mammals e.g. Homo sapiens, Mus musculus, Lepus cuniculus have been described. The mobility of proteins of blood sera has been studied over cellulose acetate paper and then a comparative pattern analysed

  13. Serology and protein electrophoresis for evidence of exposure to 12 mink pathogens in free-ranging American mink (Neovison vison) in Argentina.

    Science.gov (United States)

    Martino, Pablo Eduardo; Samartino, Luis Ernesto; Stanchi, Néstor Oscar; Radman, Nilda Esther; Parrado, Eduardo Joaquín

    2017-12-01

    Basic pathologic characteristics for farmed minks were previously reported worldwide. However, its status in the wild has not been studied in detail. Serology and electrophoresis were carried out for evidence of exposure to 12 mink pathogens on two different locations. Serology was done in 87 wild minks by reference techniques against Toxoplasma gondii, Encephalitozoon cuniculi, Neospora caninum, Brucella abortus, Mycobacterium bovis, Leptospira interrogans, canine distemper virus (CDV), canine adenovirus (CAV), canine parvovirus (CPV), rabies virus (RV), Influenza A virus (FLUAV) and Aleutian disease virus (ADV). Hypergammaglobulinemia, the ADV main clinical feature, was determined by conventional electrophoresis. Seventy-one percent of the 87 sera had antibodies against one or more pathogens. ADV accounted for the highest seroprevalence (29%), followed by T. gondii (26%), L. interrogans (14%), M. bovis (12%), B. abortus (9%), N. caninum (3%), CPV (3%) and CDV (2%). Seroprevalence was influenced by location but not sex or age. Additionally, 16% of the seropositive samples for ADV had gammaglobulin levels >40.0 g/L. Antibody titers for CDV and CPV were low and difficult to interpret as almost all these cases had borderline concentrations. A cautious interpretation of the results is urged as the epidemiological role of the wild mink is largely unexplored for most of these agents. Nevertheless, the information may be clinically relevant..

  14. Comparison of effects of age and sex on serum protein ...

    African Journals Online (AJOL)

    The aim of this study was to evaluate the influence of age and sex on the concentration of total serum protein measured by the biuret method and protein fractions determined using cellulose acetate electrophoresis in apparently healthy camels (Camelus dromedarius). Blood samples were collected from 21 camels (12 ...

  15. DNA ELECTROPHORESIS AT SURFACES

    Energy Technology Data Exchange (ETDEWEB)

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  16. Human cellular protein patterns and their link to genome DNA sequence data: usefulness of two-dimensional gel electrophoresis and microsequencing

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Leffers, H

    1991-01-01

    a global approach to the study of the cell. Using the integrated approach offered by 2-dimensional gel protein databases it is now possible to reveal phenotype specific protein (or proteins), to microsequence them, to search for homology with previously identified proteins, to clone the cDNAs, to assign...

  17. Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. I. Theoretical studies.

    Science.gov (United States)

    Hjertén, Stellan; Mohabbati, Sheila; Westerlund, Douglas

    2004-10-22

    the peaks become broad. Therefore, different types of buffers should be tested when high resolution is required. The relation sigma2 (the variance of the interaction between a protein and the buffer constituents) = constant x u (the mobility) seems to be valid for all proteins in the applied sample, at least when they have similar molecular masses. To facilitate the understanding of the progress of a free zone electrophoresis experiment, we have discussed in simple terms how the concentrations of the background electrolytes become rearranged during a run and why the difference between the mobilities of the proteins and the mobilities of the background electrolyte determines whether a peak exhibits fronting or tailing. A theoretical analysis of zone broadening in capillary zone electrophoresis, chromatography, and electrochromatography indicates that electrochromatography in homogeneous gels might be the only chromatographic technique which can compete in performance with free electrophoresis. Using an equation, valid not only for electrophoresis, but also for chromatography and centrifugation, the mobility of a concentration boundary has been calculated for the first time and was, as expected, low. Equations based on the Kohlrausch regulating function do not permit such calculations. Another regulating function (the H function) and some of its characteristics are briefly discussed. The theoretical discussions in this paper and the experimental studies in Part II show that high-performance electrophoresis deserves its prefix when the runs are designed to give minimum zone broadening. Some guidelines are given to facilitate this optimization. The plate numbers are so high that the resolution cannot be increased by more than 30% even if they approach the theoretically maximum values.

  18. Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionization time of flight mass spectroscopy combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Kehr, J; Haebel, S; Blechschmidt-Schneider, S; Willmitzer, L; Steup, M; Fisahn, J

    1999-02-01

    Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS: the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539-1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ.

  19. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, J.M. (Miami Univ., Oxford, OH (USA). Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  20. Central Gi(2) proteins, sympathetic nervous system and blood pressure regulation

    Czech Academy of Sciences Publication Activity Database

    Zicha, Josef

    2016-01-01

    Roč. 216, č. 3 (2016), s. 258-259 ISSN 1748-1708 Institutional support: RVO:67985823 Keywords : inhibitory G proteins * sympathetic nervous system * central blood pressure control Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 4.867, year: 2016

  1. Study of peripheral blood multidrug resistance-associated protein 1 expression of children intractable epilepsy.

    Science.gov (United States)

    Yue, Xuan; Liu, Xiaoming; Chen, Shengzhi; Li, Rui

    2018-04-01

    The aim of this study was to analyze multidrug resistance-associated protein 1 (MRP1) expression of peripheral blood of children intractable epilepsy. Sixty children with epilepsy admitted to outpatient and inpatient services of Xuzhou Children's Hospital between November 2010 and October 2011 were divided into a refractory epilepsy group and a drug-controlled epilepsy group, with 30 cases each. Thirty healthy children who went to the hospital in the same year for health examination were enrolled as a control group. Reverse transcriptase polymerase chain reaction and Western blot method were used to determine peripheral blood MRP1 level, mRNA, and protein content of the 3 groups. MRP1 expression in the refractory epilepsy group was significantly higher than those of the epilepsy group with good drug control and of the control group. All differences had statistical significance (P0.05). Peripheral blood MRP1 expression in patients with refractory epilepsy increases.

  2. Seed Biology of Medicinal Plants (IX) : The Relationship of Corydalis Species Derived by Gel Electrophoresis

    OpenAIRE

    米田, 該典; 加賀, 順二; 那須, 正夫; KAISUKE, YONEDA; JUNJI, KAGA; MASAO, NASU; 大阪大学薬学部; 大阪大学薬学部; 大阪大学薬学部; Faculty of Pharmaceutical Sciences, Osaka University; Faculty of Pharmaceutical Sciences, Osaka University; Faculty of Pharmaceutical Sciences, Osaka University

    1987-01-01

    The saline soluble protein fraction of seeds of the Corydalis species (Papaveraceae) in Japan was examined by polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The esterase zymogram suggested that C. pallida, C. pallida var. tenuis, C. heterocarpa var. japonica and C. speciosa, having yellow flowers and no tuber, are closely related to each other. Electrophoresis and SDS-electrophoresis patterns also coincided with the result of the esterase zymogram. They also su...

  3. Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

    Directory of Open Access Journals (Sweden)

    Li Weijun

    2011-01-01

    Full Text Available Abstract Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins, which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane

  4. Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin.

    Science.gov (United States)

    Zheng, Jianhua; Wei, Candong; Zhao, Lina; Liu, Liguo; Leng, Wenchuan; Li, Weijun; Jin, Qi

    2011-01-18

    Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions. Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work. In this

  5. Multiplexed Western Blotting Using Microchip Electrophoresis.

    Science.gov (United States)

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-05

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

  6. Sources of dietary protein in relation to blood pressure in a general Dutch population.

    Directory of Open Access Journals (Sweden)

    Wieke Altorf-van der Kuil

    Full Text Available BACKGROUND: Little is known about the relation of different dietary protein types with blood pressure (BP. We examined whether intake of total, plant, animal, dairy, meat, and grain protein was related to BP in a cross sectional cohort of 20,820 Dutch adults, aged 20-65 y and not using antihypertensive medication. DESIGN: Mean BP levels were calculated in quintiles of energy-adjusted protein with adjustment for age, sex, BMI, education, smoking, and intake of energy, alcohol, and other nutrients including protein from other sources. In addition, mean BP difference after substitution of 3 en% carbohydrates or MUFA with protein was calculated. RESULTS: Total protein and animal protein were not associated with BP (p(trend = 0.62 and 0.71 respectively, both at the expense of carbohydrates and MUFA. Systolic BP was 1.8 mmHg lower (p(trend36 g/d than in the lowest (<27 g/d quintile of plant protein. This inverse association was present both at the expense of carbohydrates and MUFA and more pronounced in individuals with untreated hypertension (-3.6 mmHg than in those with normal (+0.1 mmHg or prehypertensive BP (-0.3 mmHg; p(interaction<0.01. Meat and grain protein were not related to BP. Dairy protein was directly associated with systolic BP (+1.6 mmHg, p(trend<0.01, but not with diastolic BP (p(trend = 0.24. CONCLUSIONS: Total protein and animal protein were not associated with BP in this general untreated Dutch population. Plant protein may be beneficial to BP, especially in people with elevated BP. However, because high intake of plant protein may be a marker of a healthy diet and lifestyle in general, confirmation from randomized controlled trials is warranted.

  7. Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

    Science.gov (United States)

    Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

    2016-01-05

    A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

  8. Practical capillary electrophoresis

    CERN Document Server

    Weinberger, Robert

    2000-01-01

    In the 1980s, capillary electrophoresis (CE) joined high-performance liquid chromatography (HPLC) as the most powerful separation technique available to analytical chemists and biochemists. Published research using CE grew from 48 papers in the year of commercial introduction (1988) to 1200 in 1997. While only a dozen major pharmaceutical and biotech companies have reduced CE to routine practice, the applications market is showing real or potential growth in key areas, particularly in the DNA marketplace for genomic mapping and forensic identification. For drug development involving small molecules (including chiral separations), one CE instrument can replace 10 liquid chromatographs in terms of speed of analysis. CE also uses aqueous rather than organic solvents and is thus environmentally friendlier than HPLC. The second edition of Practical Capillary Electrophoresis has been extensively reorganized and rewritten to reflect modern usage in the field, with an emphasis on commercially available apparatus and ...

  9. Analysis of electrophoresis performance

    Science.gov (United States)

    Roberts, G. O.

    1984-01-01

    The SAMPLE computer code models electrophoresis separation in a wide range of conditions. Results are included for steady three dimensional continuous flow electrophoresis (CFE), time dependent gel and acetate film experiments in one or two dimensions and isoelectric focusing in one dimension. The code evolves N two dimensional radical concentration distributions in time, or distance down a CFE chamber. For each time or distance increment, there are six stages, successively obtaining the pH distribution, the corresponding degrees of ionization for each radical, the conductivity, the electric field and current distribution, and the flux components in each direction for each separate radical. The final stage is to update the radical concentrations. The model formulation for ion motion in an electric field ignores activity effects, and is valid only for low concentrations; for larger concentrations the conductivity is, therefore, also invalid.

  10. A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases

    DEFF Research Database (Denmark)

    Celis, J E; Madsen, Peder; Rasmussen, H H

    1991-01-01

    A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved......, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis. Udgivelsesdato: 1991-Nov...

  11. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    Science.gov (United States)

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)*

    Science.gov (United States)

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A.; Nemes, Peter

    2016-01-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. PMID:27317400

  13. Nifedipine effect on the labelling of blood cells and plasma proteins with Tc-99m

    International Nuclear Information System (INIS)

    Gutfilen, B.; Boasquevisque, E.M.; Bernardo Filho, M.

    1988-01-01

    The labeling of red blood cells (RBC) with Tc-99m depends on the presence of stannous ion (Sn) that helps this radionuclide's fixation on the hemoglobin molecule. Nifedipine is an agent capable to block a specific way where calcius (Ca) ion acrosses the cellular membrane and to bind itself on plasma proteins. The effect of nifedipine in the labeling of RBC and plasma proteins with Tc-99m was studied because of similarities between Ca and Sn ions. Blood with anticoagulant was treated with nifedipine concentration of 10 -6 M for 15 min at 37 0 C. The labeling of RBC with Tc-99m was done incubating with Sn ion solution (3 uM) for different times. The % of radioactivity in RBC was determined. Samples of plasma were precipited with trichloroacetic acid and the % of radiocctivity in insoluble fraction was calculated. The same procedure was done using different nifedipine concentrations and the blood was incubated for 60 min with Sn ion. The determination of the % of Tc-99m labeled in RBC and plasma proteins showed that this drug does not have the capability to alter this incorporation because the results are similar to control. It is suggested that the Sn ions passage across RBC is not altered by nifedipine although this drug could bind to plasma protein, it does not modify the Tc-99m fixation on it. (author) [pt

  14. Tempol protects blood proteins and lipids against peroxynitrite-mediated oxidative damage.

    Science.gov (United States)

    Mustafa, Ayman G; Bani-Ahmad, Mohammad A; Jaradat, Ahmad Q; Allouh, Mohammed Z

    2015-01-01

    Oxidative stress is characterized by excessive production of various free radicals and reactive species among which, peroxynitrite is most frequently produced in several pathological conditions. Peroxynitrite is the product of the superoxide anion reaction with nitric oxide, which is reported to take place in the intravascular compartment. Several studies have reported that peroxynitrite targets red blood cells, platelets and plasma proteins, and induces various forms of oxidative damage. This in vitro study was designed to further characterize the types of oxidative damage induced in platelets and plasma proteins by peroxynitrite. This study also determined the ability of tempol to protect blood plasma and platelets against peroxynitrite-induced oxidative damage. The ability of various concentrations of tempol (25, 50, 75, and 100 µM) to antagonize peroxynitrite-induced oxidation was evaluated by measuring the levels of protein carbonyl groups and thiobarbituric-acid-reactive substances in experimental groups. Exposure of platelets and plasma to 100 µM peroxynitrite resulted in an increased levels of carbonyl groups and lipid peroxidation (P Tempol significantly inhibited carbonyl group formation in plasma and platelet proteins (P tempol significantly reduced the levels of lipid peroxidation in both plasma and platelet samples (P tempol has antioxidative properties against peroxynitrite-induced oxidative damage in blood plasma and platelets. © 2014 by the Society for Experimental Biology and Medicine.

  15. Impact of Elevated Hemoglobin and Serum Protein on Vasovagal Reaction from Blood Donation

    Science.gov (United States)

    Tanba, Taiko; Yoshinaga, Kentaro; Motoji, Toshiko; Munakata, Masaya; Nakajima, Kazunori; Minami, Mutsuhiko

    2016-01-01

    We conducted a cross-sectional study to elucidate factors contributing to vasovagal reaction (VVR), the most frequent side effect following whole blood and apheresis donations. Complications recorded at the collection sites after voluntary donations by the Japanese Red Cross Tokyo Blood Center (JRC), in the 2006 and 2007 fiscal years, were analyzed by both univariate analysis and the multivariate conditional logistic regression model. Of 1,119,716 blood donations over the full two years, complications were recorded for 13,320 donations (1.18%), among which 67% were VVR. There were 4,303 VVR cases which had sufficient information and could be used for this study. For each VVR case, two sex- and age-matched controls (n = 8,606) were randomly selected from the donors without complications. Age, sex, body mass index (BMI), predonation blood pressure, pulse and blood test results, including total protein, albumin, and hemoglobin, were compared between the VVR group and the control group. In univariate analysis, the VVR group was significantly younger, with a lower BMI, higher blood pressure and higher blood protein and hemoglobin levels than the control group (pblood protein and hemoglobin levels showed dose-dependent relationships with VVR incidences by the Cochran-Armitage trend test (p<0.01). For both sexes, after adjusting for confounders with the multivariate conditional logistic regression model, the higher than median groups for total protein (male: OR 1.97; 95%CI 1.76,-2.21; female: OR 2.29; 95%CI 2.05–2.56), albumin (male: 1.75; 1.55–1.96; female: 1.76; 1.57–1.97) and hemoglobin (male: 1.98; 1.76–2.22; female: 1.62; 1.45–1.81) had statistically significant higher risk of VVR compared to the lower than median groups. These elevated serum protein and hemoglobin levels might offer new indicators to help understand VVR occurrence. PMID:26894814

  16. Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two-dimensional gel electrophoresis for proteomic analysis.

    Science.gov (United States)

    Nair, Sreepriya; Xavier, Tessy; Kumar, Madathiparambil Kumaran Satheesh; Saha, Sharmistha; Menon, Krishnakumar N

    2011-12-01

    Performing 2-DE of lipid-rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2-D-proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2-DE profiling of myelin proteins is very important for the detection of immuno-reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent-mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB-14-4 resulted in significant increase in protein representation of myelin in 2-DE compared with the addition of ASB-14-4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB-14-4 strategy than other strategies of myelin sample processing such as ASB-14-4 to myelin or ethanol or acetone or methanol-ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2-D gel is achieved. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. High speed capillary zone electrophoresis-mass spectrometry via an electrokinetically pumped sheath flow interface for rapid analysis of amino acids and a protein digest.

    Science.gov (United States)

    Schiavone, Nicole M; Sarver, Scott A; Sun, Liangliang; Wojcik, Roza; Dovichi, Norman J

    2015-06-01

    While capillary zone electrophoresis (CZE) has been used to produce very rapid and efficient separations, coupling these high-speed separations with mass spectrometry (MS) has been challenging. Now, with much faster and sensitive mass spectrometers, it is possible to take full advantage of the CZE speed and reconstruct the fast migrating peaks. Here are three high-speed CZE-MS analyses via an electrokinetically pumped sheath-flow interface. The first separation demonstrates CZE-ESI-MS of an amino acid mixture with a 2-min separation, >50,000 theoretical plates, low micromolar concentration detection limits, and subfemtomole mass detection limits (LTQ XL mass spectrometer). The second separation with our recently improved third-generation CE-MS interface illustrates a 20 amino acid separation in ∼7min with an average over 200,000 plate counts, and results in almost-baseline resolution of structural isomers, leucine and isoleucine. The third separation is of a BSA digest with a reproducible CZE separation and mass spectrometry detection in 2min. CZE-MS/MS analysis of the BSA digest identified 31 peptides, produced 52% sequence coverage, and generated a peak capacity of ∼40 across the 1-min separation window (Q-Exactive mass spectrometer). Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Chagas disease vector blood meal sources identified by protein mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Judith I Keller

    Full Text Available Chagas disease is a complex vector borne parasitic disease involving blood feeding Triatominae (Hemiptera: Reduviidae insects, also known as kissing bugs, and the vertebrates they feed on. This disease has tremendous impacts on millions of people and is a global health problem. The etiological agent of Chagas disease, Trypanosoma cruzi (Kinetoplastea: Trypanosomatida: Trypanosomatidae, is deposited on the mammalian host in the insect's feces during a blood meal, and enters the host's blood stream through mucous membranes or a break in the skin. Identifying the blood meal sources of triatomine vectors is critical in understanding Chagas disease transmission dynamics, can lead to identification of other vertebrates important in the transmission cycle, and aids management decisions. The latter is particularly important as there is little in the way of effective therapeutics for Chagas disease. Several techniques, mostly DNA-based, are available for blood meal identification. However, further methods are needed, particularly when sample conditions lead to low-quality DNA or to assess the risk of human cross-contamination. We demonstrate a proteomics-based approach, using liquid chromatography tandem mass spectrometry (LC-MS/MS to identify host-specific hemoglobin peptides for blood meal identification in mouse blood control samples and apply LC-MS/MS for the first time to Triatoma dimidiata insect vectors, tracing blood sources to species. In contrast to most proteins, hemoglobin, stabilized by iron, is incredibly stable even being preserved through geologic time. We compared blood stored with and without an anticoagulant and examined field-collected insect specimens stored in suboptimal conditions such as at room temperature for long periods of time. To our knowledge, this is the first study using LC-MS/MS on field-collected arthropod disease vectors to identify blood meal composition, and where blood meal identification was confirmed with more

  19. Chagas disease vector blood meal sources identified by protein mass spectrometry.

    Science.gov (United States)

    Keller, Judith I; Ballif, Bryan A; St Clair, Riley M; Vincent, James J; Monroy, M Carlota; Stevens, Lori

    2017-01-01

    Chagas disease is a complex vector borne parasitic disease involving blood feeding Triatominae (Hemiptera: Reduviidae) insects, also known as kissing bugs, and the vertebrates they feed on. This disease has tremendous impacts on millions of people and is a global health problem. The etiological agent of Chagas disease, Trypanosoma cruzi (Kinetoplastea: Trypanosomatida: Trypanosomatidae), is deposited on the mammalian host in the insect's feces during a blood meal, and enters the host's blood stream through mucous membranes or a break in the skin. Identifying the blood meal sources of triatomine vectors is critical in understanding Chagas disease transmission dynamics, can lead to identification of other vertebrates important in the transmission cycle, and aids management decisions. The latter is particularly important as there is little in the way of effective therapeutics for Chagas disease. Several techniques, mostly DNA-based, are available for blood meal identification. However, further methods are needed, particularly when sample conditions lead to low-quality DNA or to assess the risk of human cross-contamination. We demonstrate a proteomics-based approach, using liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify host-specific hemoglobin peptides for blood meal identification in mouse blood control samples and apply LC-MS/MS for the first time to Triatoma dimidiata insect vectors, tracing blood sources to species. In contrast to most proteins, hemoglobin, stabilized by iron, is incredibly stable even being preserved through geologic time. We compared blood stored with and without an anticoagulant and examined field-collected insect specimens stored in suboptimal conditions such as at room temperature for long periods of time. To our knowledge, this is the first study using LC-MS/MS on field-collected arthropod disease vectors to identify blood meal composition, and where blood meal identification was confirmed with more traditional DNA

  20. Partly replacing meat protein with soy protein alters insulin resistance and blood lipids in postmenopausal women with abdominal obesity.

    Science.gov (United States)

    van Nielen, Monique; Feskens, Edith J M; Rietman, Annemarie; Siebelink, Els; Mensink, Marco

    2014-09-01

    Increasing protein intake and soy consumption appear to be promising approaches to prevent metabolic syndrome (MetS). However, the effect of soy consumption on insulin resistance, glucose homeostasis, and other characteristics of MetS is not frequently studied in humans. We aimed to investigate the effects of a 4-wk, strictly controlled, weight-maintaining, moderately high-protein diet rich in soy on insulin sensitivity and other cardiometabolic risk factors. We performed a randomized crossover trial of 2 4-wk diet periods in 15 postmenopausal women with abdominal obesity to test diets with 22 energy percent (En%) protein, 27 En% fat, and 50 En% carbohydrate. One diet contained protein of mixed origin (mainly meat, dairy, and bread), and the other diet partly replaced meat with soy meat analogues and soy nuts containing 30 g/d soy protein. For our primary outcome, a frequently sampled intravenous glucose tolerance test (FSIGT) was performed at the end of both periods. Plasma total, LDL, and HDL cholesterol, triglycerides, glucose, insulin, and C-reactive protein were assessed, and blood pressure, arterial stiffness, and intrahepatic lipid content were measured at the start and end of both periods. Compared with the mixed-protein diet, the soy-protein diet resulted in greater insulin sensitivity [FSIGT: insulin sensitivity, 34 ± 29 vs. 22 ± 17 (mU/L)(-1) · min(-1), P = 0.048; disposition index, 4974 ± 2543 vs. 2899 ± 1878, P = 0.038; n = 11]. Total cholesterol was 4% lower after the soy-protein diet than after the mixed-protein diet (4.9 ± 0.7 vs. 5.1 ± 0.6 mmol/L, P = 0.001), and LDL cholesterol was 9% lower (2.9 ± 0.7 vs. 3.2 ± 0.6 mmol/L, P = 0.004; n = 15). Thus, partly replacing meat with soy in a moderately high-protein diet has clear advantages regarding insulin sensitivity and total and LDL cholesterol. Therefore, partly replacing meat products with soy products could be important in preventing MetS. This trial was registered at clinicaltrials

  1. Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Bak-Jensen, K.S.; Laugesen, S.; Roepstorff, P.

    2004-01-01

    gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass...

  2. Analysis of zinc oxide nanoparticles binding proteins in rat blood and brain homogenate

    Directory of Open Access Journals (Sweden)

    Shim KH

    2014-12-01

    Full Text Available Kyu Hwan Shim,1 John Hulme,1 Eun Ho Maeng,2 Meyoung-Kon Kim,3 Seong Soo A An1 1Department of Bionano Technology, Gachon Medical Research Institute, Gachon University, Sungnam-si, Gyeonggi-do, South Korea; 2Department of Analysis, KTR, Kimpo, Gyeonggi-do, South Korea; 3Department of Biochemistry and Molecular Biology, Korea University Medical School and College, Seoul, South Korea Abstract: Nanoparticles (NPs are currently used in chemical, cosmetic, pharmaceutical, and electronic products. Nevertheless, limited safety information is available for many NPs, especially in terms of their interactions with various binding proteins, leading to potential toxic effects. Zinc oxide (ZnO NPs are included in the formulation of new products, such as adhesives, batteries, ceramics, cosmetics, cement, glass, ointments, paints, pigments, and supplementary foods, resulting in increased human exposures to ZnO. Hence, we investigated the potential ZnO nanotoxic pathways by analyzing the adsorbed proteins, called protein corona, from blood and brain from four ZnO NPs, ZnOSM20(-, ZnOSM20(+, ZnOAE100(-, and ZnOAE100(+, in order to understand their potential mechanisms in vivo. Through this study, liquid chromatography–mass spectroscopy/mass spectroscopy technology was employed to identify all bound proteins. Totals of 52 and 58 plasma proteins were identified as being bound to ZnOSM20(- and ZnOSM20(+, respectively. For ZnOAE100(- and ZnOAE100(+, 58 and 44 proteins were bound, respectively. Similar numbers of proteins were adsorbed onto ZnO irrespective of size or surface charge of the nanoparticle. These proteins were further analyzed with ClueGO, a Cytoscape plugin, which provided gene ontology and the biological interaction processes of identified proteins. Interactions between diverse proteins and ZnO nanoparticles could result in an alteration of their functions, conformation, and clearance, eventually affecting many biological processes. Keywords: brain

  3. An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites.

    Science.gov (United States)

    Absalon, Sabrina; Robbins, Jonathan A; Dvorin, Jeffrey D

    2016-04-28

    Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites.

  4. Further insight into the roles of the glycans attached to human blood protein C inhibitor

    DEFF Research Database (Denmark)

    Sun, Wei; Parry, Simon; Ubhayasekera, Wimal

    2010-01-01

    Protein C inhibitor (PCI) is a 57-kDa glycoprotein that exists in many tissues and secretions in human. As a member of the serpin superfamily of proteins it displays unusually broad protease specificity. PCI is implicated in the regulation of a wide range of processes, including blood coagulation......, fertilization, prevention of tumors and pathogen defence. It has been reported that PCI isolated from human blood plasma is highly heterogeneous, and that this heterogeneity is caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of two forms that differ by the presence...... or absence of 6 amino acids at the amino-terminus. In this study we have verified that such heterogeneity exists in PCI purified from single individuals, and that individuals of two different ethnicities possess a similar PCI pattern, verifying that the micro-heterogeneity is conserved among humans...

  5. Identification of unknown protein complex members by radiolocalization and analysis of low-abundance complexes resolved using native polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Bose, Mahuya; Adams, Brian P; Whittal, Randy M; Bose, Himangshu S

    2008-02-01

    Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.

  6. Blood-based protein biomarker panel for the detection of colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Kim Y C Fung

    Full Text Available The majority of colorectal cancer (CRC cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test.In two independent cohorts (n = 145 and n = 197, we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2, Dickkopf-3 (DKK3, and Pyruvate kinase M2(PKM2.Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.

  7. Pharmaceutical protein production by yeast: towards production of human blood proteins by microbial fermentation

    OpenAIRE

    Martinez, Jose L.; Liu, Lifang; Petranovic, Dina; Nielsen, Jens

    2012-01-01

    Since the approval of recombinant insulin from Escherichia coli for its clinical use in the early 1980s, the amount of recombinant pharmaceutical proteins obtained by microbial fermentations has significantly increased. The recent advances in genomics together with high throughput analysis techniques (the so-called - omics approaches) and integrative approaches (systems biology) allow the development of novel microbial cell factories as valuable platforms for large scale production of therape...

  8. Affinity Electrophoresis for Analysis of Catalytic Module-Carbohydrate Interactions

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Svensson, Birte

    2017-01-01

    Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years, carbohy......Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years...

  9. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    Directory of Open Access Journals (Sweden)

    Tauson Anne-Helene

    2007-11-01

    Full Text Available Abstract The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets. The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver function were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively with increasing dietary BPM content, whereas the plasma glucose concentration tended to increase (P = 0.07 with increasing dietary BPM content. It was concluded that up to 50% of the nitrogen could be derived from BPM without affecting metabolic function, as reflected in the measured blood parameters.

  10. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    Science.gov (United States)

    Hellwing, Anne Louise F; Tauson, Anne-Helene; Skrede, Anders

    2007-01-01

    The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM) on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets. The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver function were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively) with increasing dietary BPM content, whereas the plasma glucose concentration tended to increase (P = 0.07) with increasing dietary BPM content. It was concluded that up to 50% of the nitrogen could be derived from BPM without affecting metabolic function, as reflected in the measured blood parameters. PMID:17996082

  11. Blood protein turnover in parasitized ruminants. The influence of host nutrition

    International Nuclear Information System (INIS)

    Dargie, J.D.

    1981-01-01

    Ruminants infected with helminth or protozoal parasites generally become anaemic and hypoalbuminaemic, as well as losing their appetite. Since feed intake plays an important part in determining blood protein levels, it is necessary, when attempting to determine the mechanisms by which parasites cause anaemia and hypoalbuminaemia, to differentiate between the effects of feed intake per se and the specific effects of the parasite on blood protein turnover. This can be done by a variety of radioisotope techniques using infected and pair-fed control animals. Additionally, animals on a poor plane of nutrition suffer more from parasitism than those which are well fed. To understand the reason for this, it is necessary to determine whether diet influences susceptibility to parasite establishment or survival, and/or susceptibility to the metabolic consequences of parasitism. Described here is the current state of knowledge on the interaction between host nutrition and susceptibility to parasitic infection and parasitic disease processes, with particular reference to anaemia and hypoalbuminaemia. It is concluded that there is little evidence that nutrition has a significant bearing on resistance or susceptibility to infection, but that it does not have a profound influence on the ability of animals to withstand the pathogenic effects of parasites. The reasons for this are discussed in detail, but the principal benefit of a good plane of nutrition is that it enables the synthetic machinery of the host to keep pace with the concurrent parasite-induced hypercatabolism of blood proteins. (author)

  12. Molecular cloning and protein structure of a human blood group Rh polypeptide

    International Nuclear Information System (INIS)

    Cherif-Zahar, B.; Bloy, C.; Le Van Kim, C.; Blanchard, D.; Bailly, P.; Hermand, P.; Salmon, C.; Cartron, J.P.; Colin, Y.

    1990-01-01

    cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO 4 /polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO 4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters

  13. Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods.

    Science.gov (United States)

    Tsao, D A; Shiau, Y F; Tseng, C S; Chang, H R

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. Materials and Mehtods: In this study, we use two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. Raf kinase inhibitor protein (RKIP), an inhibitor of Raf-mediated activation of mitogen-activated protein kinase/extracellular signal-regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time-polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non-neoplastic liver tissue of the corresponding HCC samples. These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down-regulated in liver cancer cell.

  14. Development of methods to measure hemoglobin adducts by gel electrophoresis - Preliminary results

    International Nuclear Information System (INIS)

    Sun, J.D.; McBride, S.M.

    1988-01-01

    Chemical adducts formed on blood hemoglobin may be a useful biomarker for assessing human exposures to these compounds. This paper reports preliminary results in the development of methods to measure such adducts that may be generally applicable for a wide variety of chemicals. Male F344/N rats were intraperitoneally injected with 14 C-BaP dissolved in corn oil. Twenty-four hours later, the rats were sacrificed. Blood samples were collected and globin was isolated. Globin protein was then cleaved into peptide fragments using cyanogen bromide and the fragments separated using 2-dimensional gel electrophoresis. The results showed that the adducted 14 C-globin fragments migrated to different areas of the gel than did unadducted fragments. Further research is being conducted to develop methods that will allow quantitation of separated adducted globin fragments from human blood samples without the use of a radiolabel. (author)

  15. Proteins involved in invasion of human red blood cells by malaria parasites

    Directory of Open Access Journals (Sweden)

    Ewa Jaśkiewicz

    2010-11-01

    Full Text Available Malaria is a disease caused by parasites of Plasmodium species. It is responsible for around 1-2 million deaths annually, mainly children under the age of 5. It occurs mainly in tropical and subtropical areas.Malaria is caused by five Plasmodium species:[i] P. falciparum, P. malariae, P. vivax, P. knowlesi[/i] and [i]P. ovale[/i]. Mosquitoes spread the disease by biting humans. The malaria parasite has two stages of development: the human stage and the mosquito stage. The first stage occurs in the human body and is divided into two phases: the liver phase and the blood phase.The invasion of erythrocytes by [i]Plasmodium[/i] merozoites is a multistep process of specific protein interactions between the parasite and red blood cell. The first step is the reversible merozoite attachment to the erythrocyte followed by its apical reorientation, then formation of an irreversible “tight” junction and finally entry into the red cell in a parasitophorous vacuole.The blood phase is supported by a number of proteins produced by the parasite. The merozoite surface GPI-anchored proteins (MSP-1, 2, 4, 5, 8 and 10 assist in the process of recognition of susceptible erythrocytes, apical membrane antigen (AMA-1 may be directly responsible for apical reorientation of the merozoite and apical proteins which function in tight junction formation. These ligands are members of two families: Duffy binding-like (DBL and reticulocyte binding-like (RBL proteins. In [i]Plasmodium[/i] [i]falciparum[/i] the DBL family includes: EBA-175, EBA-140 (BAEBL, EBA-181 (JESEBL, EBA-165 (PEBL and EBL-1 ligands.To date, no effective antimalarial vaccine has been developed, but there are several studies for this purpose. Therefore, it is crucial to understand the molecular basis of host cells invasion by parasites. Major efforts are focused on developing a multiantigenic and multiepitope vaccine preventing all steps of [i]Plasmodium[/i] invasion.

  16. Interaction of thorium with human blood proteins: relevance to the development of strategies for actinide decorporation

    International Nuclear Information System (INIS)

    Ali, Manjoor; Kumar, Amit; Pandey, Badri N.

    2016-01-01

    Thorium-232 (Th), a naturally-available actinide is emerging as an alternative source of nuclear fuel for energy generation in India and other countries. Therefore, understanding the biological interaction of Th and other lanthanides (Ln) related to Th fuel cycle would enable its efficient utilization with adequate human health and environmental protection. Studies on complexation behavior of Th with human blood proteins would yield a rationale insight for the development of potential chelation based strategies for decorporation of Th. Present study was carried out to characterize the site of interaction of Th/other Ln ions with the two most abundant protein of blood, human serum albumin (HSA) and hemoglobin (Hb). Using various spectroscopic techniques (UV-VIS, FT-IR, Raman, Fluorescence and Circular dichroism), results showed that Th interacts with carbonyl/amide groups of HSA and alters its secondary conformation. These effects of Th were compared with U and Ln ions. Comparing the structural/protein unfolding effects of these metals, ionic potential of metal ions seemed to determine their toxic action at protein level. In blood, Hb is the most abundant Fe-containing protein, transporting oxygen to the various parts of the body. Th and other metal ions were found to interact with peptide and heme sites of Hb depending upon the concentration. Results elucidated that the metal ions,Th and Ce(IV), which charge-ionic-radii-ratio are close to Fe(III), affected heme significantly as compared to metal ions of lower or higher charge-ionic-radii-ratio (La(III), Ce(III) and U(VI)). These results were found consistent to the effect of Th and Ce(IV) on oxygen-binding ability of Hb. In addition, deeper insight about binding characteristics of Th with HSA and Hb would have important implications for the development of decorporating agents for the biomedical management of actinides-induced toxicity. (author)

  17. Effects of a protein glycocalyx in the hemodynamics of small blood vessels

    Science.gov (United States)

    Dimakopoulos, Yiannis; Delidakis, George; Tsamopoulos, John

    2015-11-01

    Glycocalyx is a protein layer of approximate thickness 0.5 μm that lines vessel walls. We study the effects this layer has on the blood flow inside arterioles and venules, where the relative size of the glycocalyx is significant. To properly describe phenomena that naturally occur in blood flow, such as the inhomogeneous distribution of red blood cells and their aggregation, we use an improved viscoelastic constitutive model. The glycocalyx layer is modeled as fixed porous media. Cells cannot penetrate inside it, since its hydraulic permeability is very low, and the flow inside this layer is described by the equations for a viscous fluid with an extra Brinkman term to account for the effects the porous medium has on the flow. The closed set of equations is solved using the Finite Element method, assuming steady-state with dependence only in the r-direction. Our results are favorably compared with the in vivo velocity profiles in venules of mice produced by Damiano et al. (2004) and the formation of cell-free layer near glycocalyx. Flow inside the glycocalyx layer is found to be severely attenuated due to the low hydraulic permeability, which can have interesting implications in the transport of various substances form the blood to the tissues or in the use of shear stresses as signals for the endothelial surface cells. Finally, we simulate the transient blood flow under pulsatile conditions.

  18. GLUCOSE, TOTAL PROTEINS, URIC ACID AND TRIGL YCERIDES CONCENTRATIONS IN BLOOD OF ATIVE LAYING HENS

    Directory of Open Access Journals (Sweden)

    Bashir Mahmood Bhatti, Tanzeela Talat and Rozina Sardar

    2001-09-01

    Full Text Available This study was undertaken, to estimate levels of glucose, total proteins, albumins, uric acid and triglycerides in plasma of Desi and Naked Neck laying hens. The experimental birds received ration containing 16 per cent crude protein and were housed in open sheds. The mean values (mg/dL observed in Desi hens were 226.736+15.20 glucose, 1.624+0.224 albumin, 5.203+1.078 total proteins, 4.633+1.875 uric acid and 529.800+554.74 triglycerides. In Naked neck hens, the mean values were found to be 231.818+31.376 glucose, 1.562+0.287 albumins, 4.533+0.797total proteins, 4.157+1.336 uric acid and 791.200+320.474 triglycerides. There was no difference (P<0.05 in mean values of blood parameters between both the native laying hens which suggested that in identical genetical mechanism regulated concentrations of blood chemical constituents under study.

  19. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Cavalcanti, M.B.; Fernandes, T.S. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear; Amaral, A. [Universite Paris XII (UPXII) (France); Melo, J.A. [Centro de Radioterapia de Pernambuco (CERAPE), PE (Brazil); Neves, M.A.B.; Machado, C.G.F, E-mail: maribrayner@yahoo.com.br [Fundacao de Hematologia e Hemoterapia de Pernambuco, PE (Brazil)

    2005-07-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10{sub 6}/mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO{sub 2} at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results

  20. Identification of Differential Protein Expression in Hepatocellular Carcinoma Induced Wistar Albino Rats by 2D Electrophoresis and MALDI-TOF-MS Analysis.

    Science.gov (United States)

    Vedarethinam, Vadanasundari; Dhanaraj, Karthik; Soundherrajan, Ilavenil; Sivanesan, Ravikumar

    2016-04-01

    Hepato cellular carcinoma (HCC) is a type of malignant tumor. To investigate the proteins in cancer molecular mechanism and its role in HCC, we have used proteomic tools such as 2DE and MALDI-TOF-MS. Our investigation ravels that, plasma α-fetoprotein and carcinoembryonic antigen levels were elevated in DEN induced rats and gradually decreased after the treatment with 1,3BPMU. 2DE and MALDI-TOF-MS tool offers to identify the up and down regulation of proteins in HCC. Proteomic study reveals that, five differentially expressed proteins were identified in DEN induced rats and 1,3BPMU treated rats i.e. three up regulated protein such as T kininogen, NDPKB, PRMT1 (DEN induced rats), RGS19 and PAF (1,3BPMU treated rats) in 3BPMU treated rats, activation of transcription of a single gene from multiple promoters provides flexibility in the controlled gene expression. The regulations of hepatocyte stimulating factor were slow down the proliferation of hepatic cell and uncontrolled hepatic cell growth and also molecular signals strongly argue for a patho-physiological role in liver metastasis to control the cell aggression. This indicates that, anti cancer property of 1,3BPMU can be used as potent anti cancer agent. The present study also shows the proteomic approach helps to elucidate the tumor maker as well as regulatory marker proteins in HCC.

  1. Investigations on early reactions of lymphocyte proteins on gamma irradiation of human blood and their dose dependence. Preconditions for the development of an individual radiobiological dosimeter

    International Nuclear Information System (INIS)

    Turtoi, Andrei

    2007-01-01

    The present thesis is concerned with the issues involved in obtaining reliable experimental data permitting a retrospective assessment of radiation-induced doses at the time of application or contamination. In order to provide prompt medical treatment of those injured in accidents with ionizing radiation, biological procedures that can be implemented swiftly and at an early stage are required both to determine the radiation dose originally received as well as to assess the course of the dosedependent biological reactions on the basis of individual sensitivity to radiation. To this end, in the present thesis the lymphocyte proteins (phosphoproteins and total proteins) in blood taken from test subjects who had been exposed to γ-radiation (applied dose: 0-4 Gy) were analysed just 15 minutes after completing irradiation by means of 2D gel electrophoresis. Only those early-response proteins (ERPROs) that displayed a significant radiation-induced change were identified by nano-HPLC-MS/MS. For validation purposes, the dose-dependent gene expression of some of these proteins was determined by RT-qPCR. The following ERPROs displayed pronounced early reactions in the form of changes of concentration in comparison to unirradiated control samples: talin-1, talin-2, β-actin, mutant β-actin, peroxin-1 and also the phosphoproteins annexin-A6, MHCbinding protein-2, zyxin-2, interleukin-17E and phosphoglycerate kinase-1. The majority of the lymphocyte ERPROs represent proteins responsible for changes to the cytoskeleton, proliferation and cell cycle, modulation of immunoreactions as well as protein degradation and energy production. Other cellular processes may not have been determined due to the sensitivity restrictions of the 2DPAGE and MS methods, but cannot be excluded. Gene expression studies revealed that a combination of methods, comprising RT-qPCR and 2D-PAGE as well as DNA microarray and Western blot, may in future be able to overcome these restrictions. The slopes of

  2. Toxic elements and associations with hematology, plasma biochemistry, and protein electrophoresis in nesting loggerhead sea turtles (Caretta caretta) from Casey Key, Florida.

    Science.gov (United States)

    Perrault, Justin R; Stacy, Nicole I; Lehner, Andreas F; Poor, Savannah K; Buchweitz, John P; Walsh, Catherine J

    2017-12-01

    Toxic elements (arsenic, cadmium, lead, mercury, selenium, thallium) are a group of contaminants that are known to elicit developmental, reproductive, general health, and immune system effects in reptiles, even at low concentrations. Reptiles, including marine turtles, are susceptible to accumulation of toxic elements due to their long life span, low metabolic rate, and highly efficient conversion of prey into biomass. The objectives of this study were to (1) document concentrations of arsenic, cadmium, lead, mercury, selenium, and thallium in whole blood and keratin from nesting loggerhead sea turtles (Caretta caretta) from Casey Key, Florida and document correlations thereof and (2) correlate whole blood toxic element concentrations to various hematological and plasma biochemistry analytes. Baselines for various hematological and plasma analytes and toxic elements in whole blood and keratin (i.e., scute) in nesting loggerheads are documented. Various correlations between the toxic elements and hematological and plasma biochemistry analytes were identified; however, the most intriguing were negative correlations between arsenic, cadmium, lead, and selenium with and α- and γ-globulins. Although various extrinsic and intrinsic variables such as dietary and feeding changes in nesting loggerheads need to be considered, this finding may suggest a link to altered humoral immunity. This study documents a suite of health variables of nesting loggerheads in correlation to contaminants and identifies the potential of toxic elements to impact the overall health of nesting turtles, thus presenting important implications for the conservation and management of this species. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Two-dimensional fluorescence in-gel electrophoresis of coronary restenosis tissues in minipigs: increased adipocyte fatty acid binding protein induces reactive oxygen species-mediated growth and migration in smooth muscle cells.

    Science.gov (United States)

    Lu, Lin; Wang, Ya Nan; Sun, Wei Hua; Liu, Zhu Hui; Zhang, Qi; Pu, Li Jin; Yang, Ke; Wang, Ling Jie; Zhu, Zhen Bin; Meng, Hua; Yang, Ping; Du, Run; Chen, Qiu Jing; Wang, Li Shun; Yu, Hong; Shen, Wei Feng

    2013-03-01

    We aimed to uncover the protein changes of coronary artery in-stent restenosis (ISR) tissue in minipigs with and without streptozotocin-induced diabetes mellitus by quantitative 2-dimensional fluorescence in-gel electrophoresis (2D-DIGE), and to investigate the influences of crucial proteins identified, particularly adipocyte fatty acid binding protein (AFABP), in human arterial smooth muscle cells. Sirolimus-eluting stents were implanted in the coronary arteries of 15 diabetic and 26 nondiabetic minipigs, and angiography was repeated after 6 months. The intima tissue of significant ISR and non-ISR segments in both diabetic and nondiabetic minipigs was analyzed by 2D-DIGE and MALDI-TOF/TOF mass spectrometry. AFABP level was significantly increased in ISR tissue than in non-ISR tissue in both diabetic and nondiabetic minipigs, with level being higher in diabetic ISR than in nondiabetic ISR tissue. In human arterial smooth muscle cells, overexpression of AFABP significantly altered phenotype and promoted growth and migration, with effects more prominent in high-glucose than in low-glucose medium, whereas AFABP knockdown inhibited these effects. AFABP overexpression increased reactive oxygen species production by upregulating the expression of NADPH oxidase subunits Nox1, Nox4, and P22 through multiple pathways, with elevation of downstream gene cyclin D1, matrix metalloproteinase-2, and monocyte chemoattractant protein-1. However, AFABP-induced effects were inhibited by diphenyleneiodonium, pathway inhibitors, and small interfering RNA. In addition, the supernatant from AFABP-expressing human arterial smooth muscle cells and recombinant AFABP also promoted cellular growth and migration. This study has demonstrated that AFABP is significantly increased in coronary artery ISR segments of both diabetic and nondiabetic minipigs. Increased AFABP expression and secretory AFABP of human arterial smooth muscle cells promote growth and migration via reactive oxygen species

  4. Evaluation of gel electrophoresis conditions for the separation of metal-tagged proteins with subsequent laser ablation ICP-MS detection.

    Science.gov (United States)

    Raab, Andrea; Pioselli, Barbara; Munro, Caroline; Thomas-Oates, Jane; Feldmann, Jörg

    2009-01-01

    Although laser ablation (LA)-ICP-MS has been reported for the determination of metalloproteins separated by gel electrophoretic techniques (GE), systematic studies that define the conditions essential for successful measurements are still scarce. In this paper we present the results of our studies of basic conditions for the effective application of GE-LA-ICP-MS for the separation of metal-binding proteins, focusing on their stability during GE and post-separation gel treatment. The stability of metal-protein complexes (haemoglobin, myoglobin, superoxide dismutase, carbonic anhydrase, transferrin, albumin, cytochrome c) during GE is dependent on the nature of the metal-protein interaction and the principle of separation. We have observed that non-denaturing GE is a suitable separation technique for most metal-protein complexes (e.g. Zn in carbonic anhydrase and Fe in Tf and myoglobin were quantitatively recovered in a spiked liver cytosol), whereas separation by denaturing GE strongly impaired the stability of the complexes. Equally important is the post-separation treatment of the gel to enable successful detection of the metal. LA-ICP-MS requires drying of the gel without loss of protein-bound metal or cracking of the gel. This was successfully achieved using glycerol followed by heating. We demonstrate that staining of the gel prior to LA-ICP-MS using silver or Coomassie blue is not recommended, since most protein-bound metal is lost during the staining procedure. Furthermore it has been shown that only line scanning with a speed of less than 30 microm/s can reliably distinguish between lines 1 mm apart, while raster spot analysis carries the risk of misinterpretation due to contamination in/on inhomogeneous gels.

  5. Regulator of G-protein signaling 5 controls blood pressure homeostasis and vessel wall remodeling.

    Science.gov (United States)

    Holobotovskyy, Vasyl; Manzur, Mitali; Tare, Marianne; Burchell, Jennifer; Bolitho, Erin; Viola, Helena; Hool, Livia C; Arnolda, Leonard F; McKitrick, Douglas J; Ganss, Ruth

    2013-03-01

    Regulator of G-protein signaling 5 (RGS5) modulates G-protein-coupled receptor signaling and is prominently expressed in arterial smooth muscle cells. Our group first reported that RGS5 is important in vascular remodeling during tumor angiogenesis. We hypothesized that RGS5 may play an important role in vessel wall remodeling and blood pressure regulation. To demonstrate that RGS5 has a unique and nonredundant role in the pathogenesis of hypertension and to identify crucial RGS5-regulated signaling pathways. We observed that arterial RGS5 expression is downregulated with chronically elevated blood pressure after angiotensin II infusion. Using a knockout mouse model, radiotelemetry, and pharmacological inhibition, we subsequently showed that loss of RGS5 results in profound hypertension. RGS5 signaling is linked to the renin-angiotensin system and directly controls vascular resistance, vessel contractility, and remodeling. RGS5 deficiency aggravates pathophysiological features of hypertension, such as medial hypertrophy and fibrosis. Moreover, we demonstrate that protein kinase C, mitogen-activated protein kinase/extracellular signal-regulated kinase, and Rho kinase signaling pathways are major effectors of RGS5-mediated hypertension. Loss of RGS5 results in hypertension. Loss of RGS5 signaling also correlates with hyper-responsiveness to vasoconstrictors and vascular stiffening. This establishes a significant, distinct, and causal role of RGS5 in vascular homeostasis. RGS5 modulates signaling through the angiotensin II receptor 1 and major Gαq-coupled downstream pathways, including Rho kinase. So far, activation of RhoA/Rho kinase has not been associated with RGS molecules. Thus, RGS5 is a crucial regulator of blood pressure homeostasis with significant clinical implications for vascular pathologies, such as hypertension.

  6. Does whey protein supplementation affect blood pressure in hypoalbuminemic peritoneal dialysis patients?

    Directory of Open Access Journals (Sweden)

    Hassan K

    2017-08-01

    Full Text Available Kamal Hassan,1,2 Fadi Hassan3 1Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, 2Department of Nephrology and Hypertension, Peritoneal Dialysis Unit, 3Department of Internal Medicine E, Galilee Medical Center, Nahariya, Israel Objective: Hypertension and hypoalbuminemia are common risk factors for cardiovascular complications in peritoneal dialysis (PD patients. Data are limited regarding the effects of whey protein consumption on blood pressure in this population. The aim of the present study was to examine if whey protein supplementation for 12 weeks to hypoalbuminemic PD patients affects their blood pressure.Patients and methods: This prospective randomized study included 36 stable PD patients with serum albumin levels <3.8 g/dL. During 12 weeks, 18 patients were instructed to consume 1.2 g/kg/day of protein and an additional whey protein supplement at a dose of 25% of the instructed daily protein (whey protein group. Eighteen patients were instructed to consume protein in the amount of 1.2 g/kg/day and an additional 25%, without whey protein supplementation (control group. Results: Compared to the control group, in the whey protein group, serum albumin levels, oncotic pressure, and dialysate ultrafiltration significantly increased (3.55±0.14 to 4.08±0.15 g/dL, P<0.001; 21.81±2.03 to 24.06±1.54 mmHg, P<0.001; 927.8±120.3 to 1,125.0±125.1 mL/day, P<0.001; respectively and were significantly higher after 12 weeks (4.08±0.15 vs 3.41±0.49 g/dL, P<0.001; 24.06±1.54 vs 22.71±1.77 mmHg, P=0.010; 1,125.0±125.1 vs 930.6±352.8 mL/day, P=0.017; respectively in the whey protein group compared to the control group. Fluid overload, the extracellular to intracellular ratio and mean arterial pressure (MAP significantly decreased (2.46±1.08 to 1.52±0.33, P<0.001; 1.080±0.142 to 0.954±0.124, P<0.001; 102.6±3.80 to 99.83±3.85, P=0.018; respectively and were significantly lower in the whey protein group after 12 weeks (1.52±0

  7. Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

    Science.gov (United States)

    Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

    2016-02-01

    Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

  8. Blood responses of calves fed milk substitutes containing hydrolyzed fish protein and lime-treated corn flour.

    Science.gov (United States)

    Diaz-Castaneda, M; Brisson, G J

    1989-08-01

    Two experiments involving 3- to 5-d-old dairy calves were carried out. In Experiment 1, lime-treated corn flour (Nixtamal) supplied 50 to 100% of carbohydrates in a milk substitute based on sodium caseinate, lard, and cerelose. In Experiment 2, partially hydrolyzed fish protein concentrate replaced 50% of 67% of proteins in milk substitutes based on skim milk powder, lard, and 35% Nixtamal. Increasing the proportion of carbohydrates supplied by Nixtamal was associated with a linear decrease of postprandial serum glucose and insulin. Postprandial fluctuations in blood glucose were less in calves fed Nixtamal than in controls. Nixtamal probably was trapped within the casein clot in the abomasum, leading to delayed rate of passage of Nixtamal carbohydrates into the intestine. Replacing skim milk protein with hydrolyzed fish protein in diets containing Nixtamal had no effect on blood glucose or insulin but elevated free essential amino acids, which promoted glucagon secretion. More uniform concentrations of blood essential amino acids and glucose were related to lower blood urea at 54 d in calves fed diets based on hydrolyzed fish protein and Nixtamal, than that of control calves. It is suggested that more uniform postprandial blood glucose concentrations might reduce amino acid degradation for energy purposes and stimulate protein synthesis. Young dairy calves may adapt to milk substitutes based on Nixtamal and hydrolyzed fish protein despite changes in the concentration patterns of several blood components.

  9. Blood

    Science.gov (United States)

    ... production of red blood cells, including: Iron deficiency anemia. Iron deficiency anemia is the most common type of anemia and ... inflammatory bowel disease are especially likely to have iron deficiency anemia. Anemia due to chronic disease. People with chronic ...

  10. Analyses of cardiac blood cells and serum proteins with regard to cause of death in forensic autopsy cases.

    Science.gov (United States)

    Quan, Li; Ishikawa, Takaki; Michiue, Tomomi; Li, Dong-Ri; Zhao, Dong; Yoshida, Chiemi; Chen, Jian-Hua; Komatsu, Ayumi; Azuma, Yoko; Sakoda, Shigeki; Zhu, Bao-Li; Maeda, Hitoshi

    2009-04-01

    To investigate hematological and serum protein profiles of cadaveric heart blood with regard to the cause of death, serial forensic autopsy cases (n=308, >18 years of age, within 48 h postmortem) were examined. Red blood cells (Rbc), hemoglobin (Hb), platelets (Plt), white blood cells (Wbc), total protein (TP) and albumin (Alb) were examined in bilateral cardiac blood. Blood cell counts, collected after turning the bodies at autopsy, approximated to the clinical values. Postmortem changes were not significant for these markers. In non-head blunt injury cases, Rbc counts, Hb, TP and Alb levels in bilateral cardiac blood were lower in subacute deaths (survival time, 1-12 h) than in acute deaths (survival time hematology analyzer than by using a blood smear test, suggesting Rbc fragmentation caused by deep burns, while increases in Wbc count and decreases in Alb levels were seen for subacute deaths. For asphyxiation, Rbc count, Hb, TP and Alb levels in bilateral cardiac blood were higher than other groups, and TP and Alb levels in the right cardiac blood were higher for hanging than for strangulation. These findings suggest that analyses of blood cells and proteins are useful for investigating the cause of death.

  11. Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

    DEFF Research Database (Denmark)

    Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V

    1998-01-01

    Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI...

  12. Paleoproteomics explained to youngsters: how did the wedding of two-dimensional electrophoresis and protein sequencing spark proteomics on: let there be light.

    Science.gov (United States)

    Rabilloud, Thierry

    2014-07-31

    Taking the opportunity of the 20th anniversary of the word "proteomics", this young adult age is a good time to remember how proteomics came from enormous progress in protein separation and protein microanalysis techniques, and from the conjugation of these advances into a high performance and streamlined working setup. However, in the history of the almost three decades that encompass the first attempts to perform large scale analysis of proteins to the current high throughput proteomics that we can enjoy now, it is also interesting to underline and to recall how difficult the first decade was. Indeed when the word was cast, the battle was already won. This recollection is mostly devoted to the almost forgotten period where proteomics was being conceived and put to birth, as this collective scientific work will never appear when searched through the keyword "proteomics". The significance of this manuscript is to recall and review the two decades that separated the first attempts of performing large scale analysis of proteins from the solid technical corpus that existed when the word "proteomics" was coined twenty years ago. This recollection is made within the scientific historical context of this decade, which also saw the blossoming of DNA cloning and sequencing. This article is part of a Special Issue entitled: 20 years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini , Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Arterial Blood Pressure Induces Transient C4b-Binding Protein in Human Saphenous Vein Grafts.

    Science.gov (United States)

    Kupreishvili, Koba; Meischl, Christof; Vonk, Alexander B A; Stooker, Wim; Eijsman, Leon; Blom, Anna M; Quax, Paul H A; van Hinsbergh, Victor W M; Niessen, Hans W M; Krijnen, Paul A J

    2017-05-01

    Complement is an important mediator in arterial blood pressure-induced vein graft failure. Previously, we noted activation of cell protective mechanisms in human saphenous veins too. Here we have analyzed whether C4b-binding protein (C4bp), an endogenous complement inhibitor, is present in the vein wall. Human saphenous vein segments obtained from patients undergoing coronary artery bypass grafting (n = 55) were perfused in vitro at arterial blood pressure with either autologous blood for 1, 2, 4, or 6 hr or with autologous blood supplemented with reactive oxygen species scavenger N-acetylcysteine. The segments were subsequently analyzed quantitatively for presence of C4bp and complement activation product C3d using immunohistochemistry. Perfusion induced deposition of C3d and C4bp within the media of the vessel wall, which increased reproducibly and significantly over a period of 4 hr up to 3.8% for C3d and 81% for C4bp of the total vessel area. Remarkably after 6 hr of perfusion, the C3d-positive area decreased significantly to 1.3% and the C4bp-positive area to 19% of the total area of the vein. The areas positive for both C4bp and C3d were increased in the presence of N-acetylcysteine. Exposure to arterial blood pressure leads to a transient presence of C4bp in the vein wall. This may be part of a cell-protective mechanism to counteract arterial blood pressure-induced cellular stress and inflammation in grafted veins. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Comparative Two-Dimensional Fluorescence Gel Electrophoresis.

    Science.gov (United States)

    Ackermann, Doreen; König, Simone

    2018-01-01

    Two-dimensional comparative fluorescence gel electrophoresis (CoFGE) uses an internal standard to increase the reproducibility of coordinate assignment for protein spots visualized on 2D polyacrylamide gels. This is particularly important for samples, which need to be compared without the availability of replicates and thus cannot be studied using differential gel electrophoresis (DIGE). CoFGE corrects for gel-to-gel variability by co-running with the sample proteome a standardized marker grid of 80-100 nodes, which is formed by a set of purified proteins. Differentiation of reference and analyte is possible by the use of two fluorescent dyes. Variations in the y-dimension (molecular weight) are corrected by the marker grid. For the optional control of the x-dimension (pI), azo dyes can be used. Experiments are possible in both vertical and horizontal (h) electrophoresis devices, but hCoFGE is much easier to perform. For data analysis, commercial software capable of warping can be adapted.

  15. Aluminum complexing enhances amyloid beta protein penetration of blood-brain barrier.

    Science.gov (United States)

    Banks, William A; Niehoff, Michael L; Drago, Denise; Zatta, Paolo

    2006-10-20

    A significant co-morbidity of Alzheimer's disease and cerebrovascular impairment suggests that cerebrovascular dysregulation is an important feature of dementia. Amyloid beta protein (Abeta), a relevant risk factor in Alzheimer's disease, has neurotoxic properties and is thought to play a critical role in the cognitive impairments. Previously, we demonstrated that the 42mer of Abeta (Abeta42) complexed with aluminum (Al-Abeta42) is much more cytotoxic than non-complexed Abeta42. The level of Abeta in the brain is a balance between synthesis, degradation, and fluxes across the blood-brain barrier (BBB). In the present paper, we determined whether complexing with aluminum affected the ability of radioactively iodinated Abeta to cross the in vivo BBB. We found that the rates of uptake of Al-Abeta42 and Abeta42 were similar, but that Al-Abeta42 was sequestered by brain endothelial cells much less than Abeta42 and so more readily entered the parenchymal space of the brain. Al-Abeta42 also had a longer half-life in blood and had increased permeation at the striatum and thalamus. Brain-to-blood transport was similar for Al-Abeta42 and Abeta42. In conclusion, complexing with aluminum affects some aspects of blood-to-brain permeability so that Al-Abeta42 would have more ready access to brain cells than Abeta42.

  16. Rapid capillary electrophoresis approach for the quantification of ewe milk adulteration with cow milk.

    Science.gov (United States)

    Trimboli, Francesca; Morittu, Valeria Maria; Cicino, Caterina; Palmieri, Camillo; Britti, Domenico

    2017-10-13

    The substitution of ewe milk with more economic cow milk is a common fraud. Here we present a capillary electrophoresis method for the quantification of ewe milk in ovine/bovine milk mixtures, which allows for the rapid and inexpensive recognition of ewe milk adulteration with cow milk. We utilized a routine CE method for human blood and urine proteins analysis, which fulfilled the separation of skimmed milk proteins in alkaline buffer. Under this condition, ovine and bovine milk exhibited a recognizable and distinct CE protein profiles, with a specific ewe peak showing a reproducible migration zone in ovine/bovine mixtures. Based on ewe specific CE peak, we developed a method for ewe milk quantification in ovine/bovine skimmed milk mixtures, which showed good linearity, precision and accuracy, and a minimum amount of detectable fraudulent cow milk equal to 5%. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Blood serum components and serum protein test of Hybro-PG broilers of different ages

    Directory of Open Access Journals (Sweden)

    PRL Silva

    2007-12-01

    Full Text Available Blood serum samples of HYBRO PG broilers were analyzed, with 30 samples collected from 21-day-old broilers (G1, 30 from 35-day-old birds (G2, and 30 from 42-day-old birds (G3, with the aim of establishing normal values of some blood serum parameters. The activities of the enzymes gamma-glutamyl-transferase (GGT, aspartate aminotransferase (AST, creatine kinase (CK, alkaline phosphatase (ALP, and lactate dehydrogenase (LDH, serum levels of total calcium, calcium ion, phosphorus, sodium, potassium, magnesium, chlorides, creatinine, uric acid, triglycerides, cholesterol, total protein, albumin, total and indirect and direct bilirubin, and electrophoretic profile of serum proteins in acrylamide (SDS-PAGE and agarose gel were determined. There was no influence of age on total bilirubin and albumin levels. All the other evaluated parameters presented differences in at least one age group. Protein electrophoretic profile also changed as a function of age. The obtained results can be considered as normal for the studied ages, and therefore be used as references for the interpretation of laboratory exams of broilers of this genetic line in the evaluated ages.

  18. alpha isoforms of soluble and membrane-linked folate-binding protein in human blood

    DEFF Research Database (Denmark)

    Hoier-Madsen, M.; Holm, J.; Hansen, S.I.

    2008-01-01

    supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were established for the first time in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes......, and in granulocytes and serum, only constituted an almost undetectable fraction of the functional FBP The FBP alpha in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of FBP alpha (>120 kDa), which could represent receptor...... fragments from decomposed erythrocytes and granulocytes. The soluble FBPs may exert bacteriostatic effects and protect folates in plasma from biological degradation, whereas FRs on the surface of blood cells could be involved in intracellular folate uptake or serve as signal proteins. The latter receptors...

  19. Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus.

    Science.gov (United States)

    Chen, X; Threlkeld, S W; Cummings, E E; Juan, I; Makeyev, O; Besio, W G; Gaitanis, J; Banks, W A; Sadowska, G B; Stonestreet, B S

    2012-12-13

    The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (Pbrain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4 h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4 h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Identification of Salivary Gland Proteins Depleted after Blood Feeding in the Malaria Vector Anopheles campestris-like Mosquitoes (Diptera: Culicidae)

    OpenAIRE

    Sor-suwan, Sriwatapron; Jariyapan, Narissara; Roytrakul, Sittiruk; Paemanee, Atchara; Phumee, Atchara; Phattanawiboon, Benjarat; Intakhan, Nuchpicha; Chanmol, Wetpisit; Bates, Paul A.; Saeung, Atiporn; Choochote, Wej

    2014-01-01

    Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles ca...

  1. Phylogenetic analyses and detection of viridans streptococci based on sequences and denaturing gradient gel electrophoresis of the rod shape-determining protein gene

    OpenAIRE

    Konishi, Ikuri; Hoshino, Tomonori; Kondo, Yoshio; Saito, Kan; Nishiguchi, Miyuki; Sato, Kyoko; Fujiwara, Taku

    2009-01-01

    Background: Population analysis of viridans streptococci is important because these species are associated with dental caries, bacteremia, and subacute endocarditis, in addition to being important members of the human oral commensal microbiota. Design: In this study, we phylogenetically analyzed the rod shape-determining protein gene (rodA), which is associated with cellular morphology, cell division, and sensitivity for antibiotics, and demonstrated that the diversity of the rodA gene is suf...

  2. Identification of Proteins Modulated in the Date Palm Stem Infested with Red Palm Weevil (Rhynchophorus ferrugineus Oliv.) Using Two Dimensional Differential Gel Electrophoresis and Mass Spectrometry.

    Science.gov (United States)

    Rasool, Khawaja Ghulam; Khan, Muhammad Altaf; Aldawood, Abdulrahman Saad; Tufail, Muhammad; Mukhtar, Muhammad; Takeda, Makio

    2015-08-17

    A state of the art proteomic methodology using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI TOF) has been employed to characterize peptides modulated in the date palm stem subsequent to infestation with red palm weevil (RPW). Our analyses revealed 32 differentially expressed peptides associated with RPW infestation in date palm stem. To identify RPW infestation associated peptides (I), artificially wounded plants (W) were used as additional control beside uninfested plants, a conventional control (C). A constant unique pattern of differential expression in infested (I), wounded (W) stem samples compared to control (C) was observed. The upregulated proteins showed relative fold intensity in order of I > W and downregulated spots trend as W > I, a quite interesting pattern. This study also reveals that artificially wounding of date palm stem affects almost the same proteins as infestation; however, relative intensity is quite lower than in infested samples both in up and downregulated spots. All 32 differentially expressed spots were subjected to MALDI-TOF analysis for their identification and we were able to match 21 proteins in the already existing databases. Relatively significant modulated expression pattern of a number of peptides in infested plants predicts the possibility of developing a quick and reliable molecular methodology for detecting plants infested with date palm.

  3. Effect of soya protein on blood pressure: a meta-analysis of randomised controlled trials.

    Science.gov (United States)

    Dong, Jia-Yi; Tong, Xing; Wu, Zhi-Wei; Xun, Peng-Cheng; He, Ka; Qin, Li-Qiang

    2011-08-01

    Observational studies have indicated that soya food consumption is inversely associated with blood pressure (BP). Evidence from randomised controlled trials (RCT) on the BP-lowering effects of soya protein intake is inconclusive. We aimed to evaluate the effectiveness of soya protein intake in lowering BP. The PubMed database was searched for published RCT in the English language through to April 2010, which compared a soya protein diet with a control diet. We conducted a random-effects meta-analysis to examine the effects of soya protein on BP. Subgroup and meta-regression analyses were performed to explore possible explanations for heterogeneity among trials. Meta-analyses of twenty-seven RCT showed a mean decrease of 2·21 mmHg (95 % CI - 4·10, - 0·33; P = 0·021) for systolic BP (SBP) and 1·44 mmHg (95 % CI - 2·56, - 0·31; P = 0·012) for diastolic BP (DBP), comparing the participants in the soya protein group with those in the control group. Soya protein consumption significantly reduced SBP and DBP in both hypertensive and normotensive subjects, and the reductions were markedly greater in hypertensive subjects. Significant and greater BP reductions were also observed in trials using carbohydrate, but not milk products, as the control diet. Meta-regression analyses further revealed a significantly inverse association between pre-treatment BP and the level of BP reductions. In conclusion, soya protein intake, compared with a control diet, significantly reduces both SBP and DBP, but the BP reductions are related to pre-treatment BP levels of subjects and the type of control diet used as comparison.

  4. Ontogeny and characterization of blood leukocyte subsets and serum proteins in piglets before and after weaning

    DEFF Research Database (Denmark)

    Juul-Madsen, H.R.; Jensen, K.H.; Nielsen, Jens

    2010-01-01

    cytometry using monoclonal antibodies against CD1, CD3, CD4, CD8a CD14, CD21, CD172 (SWC3a), CD284 (TLR4), SLAI, and SLAII were performed to identify T-lymphocyte subsets, B-lymphocytes, monocytes, and granulocytes. ELISA was used to measure the concentration of serum proteins. Several of the analyzed......Existing knowledge about the development of the porcine immune system was extended by phenotypic characterization of leukocyte subsets and with assessment of Mannan-Binding Lectin (MBL) and immunoglobulin concentrations in peripheral blood of healthy piglets. Single-color and/or double-color flow...

  5. Influence of alfalfa protein concentrate dietary supplementation on blood parameters of growing-finishing pigs

    Directory of Open Access Journals (Sweden)

    Pietrzak Krzysztof

    2015-09-01

    Full Text Available The aim of the study was to analyse the effect of the feed additive alfalfa protein concentrate (APC, on pig health. The trial involved 40 crossbred gilts and 40 crossbred castrates (Polish Landrace × Polish Large White × Duroc of 29.0 ± 0.5 kg initial body weight. Allocation of experimental animals was into four treatment groups: the control group (C was fed standard mixtures, without APC addition; group E-15 was fed a basal diet supplemented with 1.5% APC; and groups E-30 and E-30P were fed diets with 3.0% APC inclusion. There were two feeding systems. In the first system, animals of groups C, E-15, and E-30 were fed continuously with suitable mixtures. The second feeding system was used in group E-30P where animals received the experimental or control mixture alternating at two-week intervals. The addition of APC supplement to diets significantly increased (P ≤ 0.05 red blood cell indices, i.e. haematocrit (Ht, red blood cell count (RBC, and haemoglobin concentration (Hb in growing and finishing periods. The analysis of enzyme activity demonstrated a markedly higher activity of aspartate aminotransferase (AST, alanine aminotransferase (ALT, and especially alkaline phosphatase (ALP in the blood plasma of pigs fed APC supplement. This increase may indicate a negative impact of APC on the animal’s liver. A positive effect of dietary APC on blood lipid parameters was associated with a decreased level of total cholesterol and reduced low-density lipoprotein fraction. Analysis of the haematological and biochemical blood indices demonstrated that APC additive may affect animal health.

  6. Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding.

    Directory of Open Access Journals (Sweden)

    Tae Kwon Kim

    2016-01-01

    Full Text Available Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD, and spontaneously detached (SD. The 582 tick proteins include proteases (5.7%, protease inhibitors (7.4%, unknown function proteins (22%, immunity/antimicrobial (2.6%, lipocalin (3.1%, heme/iron binding (2.6%, extracellular matrix/ cell adhesion (2.2%, oxidant metabolism/ detoxification (6%, transporter/ receptor related (3.2%, cytoskeletal (5.5%, and housekeeping-like (39.7%. Notable observations include: (i tick saliva proteins of unknown function accounting for >33% of total protein content, (ii 79% of proteases are metalloproteases, (iii 13% (76/582 of proteins in this study were found in saliva of other tick species and, (iv ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the tick's antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the tick's strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick

  7. Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding

    Science.gov (United States)

    Pinto, Antônio F. M.; Moresco, James; Yates, John R.; da Silva Vaz, Itabajara; Mulenga, Albert

    2016-01-01

    Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD) agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD), and spontaneously detached (SD). The 582 tick proteins include proteases (5.7%), protease inhibitors (7.4%), unknown function proteins (22%), immunity/antimicrobial (2.6%), lipocalin (3.1%), heme/iron binding (2.6%), extracellular matrix/ cell adhesion (2.2%), oxidant metabolism/ detoxification (6%), transporter/ receptor related (3.2%), cytoskeletal (5.5%), and housekeeping-like (39.7%). Notable observations include: (i) tick saliva proteins of unknown function accounting for >33% of total protein content, (ii) 79% of proteases are metalloproteases, (iii) 13% (76/582) of proteins in this study were found in saliva of other tick species and, (iv) ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the tick's antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the tick's strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick

  8. Establishment of the Optimal Conditions for Two–Dimensional Gel Electrophoresis of Papaya Seed Proteome

    OpenAIRE

    Roan, Su–Feng; Hsiung, Tung–Chuan; Yang, Kai–Yun; Liu, Wei–Ting; Chang, Ing–Feng; Wu, Chun–Ta; Wakana, Akira; 若菜, 章

    2013-01-01

    Papaya seeds are considered as a recalcitrant material for protein preparation in two-dimensional gel electrophoresis (2-DE) because of abundance of interfering compounds. We examined two protein extraction methods, the classical trichloroacetic acid (TCA)

  9. Polyacrylamide gel electrophoresis of RNA.

    Science.gov (United States)

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W

    2010-06-01

    Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. This technique is generally applicable for RNA detection, quantification, purification by size, and quality assessment. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to RNA-protein complexes, native gels are appropriate. The only disadvantage to acrylamide gels is that they are not suitable for analyzing large RNAs (> or =600 nt); for such applications, agarose gels are preferred. This protocol describes how to prepare, load, and run polyacrylamide gels for RNA analysis.

  10. SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

    Science.gov (United States)

    Taylor, Shannon; Sunshine, Sara S.; McCarthy, Kathleen; Risher, Nicole; Newcomb, Tara; Weetall, Marla; Prior, Thomas W.; Swoboda, Kathryn J.; Chen, Karen S.; Paushkin, Sergey

    2016-01-01

    Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy. PMID:26953792

  11. Myeloperoxidase activity and the oxidized proteins in blood neutrophils of patients with pneumonia.

    Science.gov (United States)

    Muravlyova, Larissa; Molotov-Luchanskiy, Vilen; Bakirova, Ryszhan; Klyuyev, Dmitriy; Demidchik, Ludmila; Kolesnikova, Yevgeniya

    2014-10-01

    The main purpose of our investigation was to study myeloperoxidase activity and concentration of oxidized proteins in blood neutrophils of patients with ambulant pneumonia and secondary pneumonia which has arisen on a background of chronic obstructive pulmonary disease (COPD). Patients were divided into 2 groups. 17 patients with ambulant pneumonia moderate severity and respiratory insufficiency of grade 2 were included in the 1-st group. 20 COPD patients with secondary pneumonia moderate severity and with respiratory insufficiency of grade 2 were included in the 2-nd group. The control group consisted of 15 healthy subjects. The reactive protein carbonyl derivates, advanced oxidation protein products (AOPP) and myeloperoxidase activity were detected in neutrophils. In neutrophils of 1-st group patients the augmentation of reactive protein carbonyl derivates was observed in comparison with healthy ones. In neutrophils of 2-nd group patients the slight decrease of reactive protein carbonyl derivates was observed in comparison with healthy ones (by 17%). In neutrophils of 2-nd group patients the significant increasing AOPP in comparison with healthy ones (p <0.01) and 1 group patients (p <0.05) was fixed. Myeloperoxidase activity was higher in neutrophils of 1-th group patients in comparison with healthy ones. In neutrophils of 2-nd group patients myeloperoxidase activity was higher in comparison with the same of 1 group patients (by 67%, p <0.05). Our results showed the different direction of oxidized proteins formation neutrophils of patients with primary and secondary pneumonia. Besides that the varied degree of myeloperoxidase activity was fixed. Our results require more detailed understanding because they can reflect peculiar mechanisms of pneumonia development and determine the characteristics of their progression. Copyright © 2014. Published by Elsevier Inc.

  12. THE ROLE OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN (MRP) IN THE BLOOD-BRAIN BARRIER AND OPIOID ANALGESIA

    Science.gov (United States)

    Su, Wendy; Pasternak, Gavril W.

    2013-01-01

    The blood brain barrier protects the brain from circulating compounds and drugs. The ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) is involved with the barrier, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood, raising the possibility of a similar role for other transporters. Multidrug resistance associated protein (MRP), a 190 kDa protein similar to Pgp is also ABC transport that has been implicated in the blood brain barrier. The current study explores its role in opioid action. Immunohistochemically, it is localized in the choroid plexus in ratsand can be selectively downregulated by antisense treatment at both the level of mRNA, as shown by RT-PCR, and protein, as demonstrated immunohistochemically. Behaviorally, downregulation of MRP significantly enhances the analgesic potency of systemic morphine in MRP knockout mice and in antisense-treated rats by lowering the blood brain barrier. Following intracerebroventricular administration, a number of compounds, including some opioids, are rapidly secreted from the brain into the blood where they contribute to the overall analgesic effects by activating peripheral systems. MRP plays a role in this efflux. Downregulating MRP expression leads to a corresponding decrease in the transport and a diminished analgesic response from opioids administered intracerebroventricularly. Thus, the transporter protein MRP plays a role in maintaining the blood-brain barrier and modulates the activity of opioids. PMID:23508590

  13. Artificial neural networks to model the production of blood protein hydrolysates for plant fertilisation.

    Science.gov (United States)

    Gálvez, Raúl Pérez; Carpio, Francisco Javier Espejo; Guadix, Emilia M; Guadix, Antonio

    2016-01-15

    Amino acid-based fertilisers increase the bioavailability of nitrogen in plants and help withstand stress conditions. Additionally, porcine blood protein hydrolysates are able to supply iron, which is involved in chlorophyll synthesis and improves the availability of nutrients in soil. A high degree of hydrolysis is desirable when producing a protein hydrolysate intended for fertilisation, since it assures a high supply of free amino acids. Given the complexity of enzyme reactions, empirical approaches such as artificial neural networks (ANNs) are preferred for modelisation. Porcine blood meal was hydrolysed for 3 h with subtilisin. The time evolution of the degree of hydrolysis was successfully modelled by means of a feedforward ANN comprising 10 neurons in the hidden layer and trained by the Levenberg-Marquardt algorithm. The ANN model described adequately the influence of pH, temperature, enzyme concentration and reaction time upon the degree of hydrolysis, and was used to estimate the optimal operation conditions (pH 6.67, 56.9 °C, enzyme to substrate ratio of 10 g kg(-1) and 3 h of reaction) leading to the maximum degree of hydrolysis (35.12%). ANN modelling was a useful tool to model enzymatic reactions and was successfully employed to optimise the degree of hydrolysis. © 2015 Society of Chemical Industry.

  14. Optimization of capillary electrophoresis?inductively coupled plasma mass spectrometry for species analysis of metallothionein-like proteins extracted from liver tissues of Elbe-bream and Roe deer

    Science.gov (United States)

    Pröfrock, Daniel; Prange, Andreas; Schaumlöffel, Dirk; Ruck, Wolfgang

    2003-08-01

    Species analysis of metallothionein-like proteins (MLP) in liver tissues from Elbe-Bream ( Abramis brama L.) and Roe Deer ( Capreolus capreolus L.) using capillary electrophoresis (CE) combined with inductively coupled plasma mass spectrometry detection is described. In order to allow systematic development of the method, commercially available metallothionein (MT) preparations of rabbit liver were used. Optimum separation efficiency was obtained by investigating the influence of parameters such as voltage, capillary temperature, buffer concentration, buffer pH and the use of different buffer systems. Instrumental parameters such as CE capillary position, interface adjustment and contamination problems are also discussed. Separation was performed using uncoated fused silica capillaries with 75 μm i.d. and 70 cm length. The optimum conditions were found to be: Separation voltage 30 kV, positive polarity, capillary temperature 288.15 K and a buffer concentration of 100 mmol l -1 Tricine-NH 3 adjusted to pH 7.2. Sample preparation was performed so as to minimize oxidation and heavy metal contamination of the samples. The high molecular mass protein matrix was reduced by acetonitrile precipitation. For commercial MT preparations the relative standard deviations (R.S.D) in the retention times were 0.9% for MT-1 and 1.9% for MT-2; the R.S.D.'s in the peak areas were less than 6% for MT-1 and 16% for MT-2, respectively. Under optimized conditions the MLPs in the real samples could be separated efficiently in less than 10 min. By comparison with the migration times of commercially available MT preparations, two of the observed peaks could be assigned to MT-1 and MT-2.

  15. Optimization of capillary electrophoresis-inductively coupled plasma mass spectrometry for species analysis of metallothionein-like proteins extracted from liver tissues of Elbe-bream and Roe deer

    Energy Technology Data Exchange (ETDEWEB)

    Proefrock, Daniel; Prange, Andreas E-mail: andreas.prange@gkss.de; Schaumloeffel, Dirk; Ruck, Wolfgang

    2003-08-15

    Species analysis of metallothionein-like proteins (MLP) in liver tissues from Elbe-Bream (Abramis brama L.) and Roe Deer (Capreolus capreolus L.) using capillary electrophoresis (CE) combined with inductively coupled plasma mass spectrometry detection is described. In order to allow systematic development of the method, commercially available metallothionein (MT) preparations of rabbit liver were used. Optimum separation efficiency was obtained by investigating the influence of parameters such as voltage, capillary temperature, buffer concentration, buffer pH and the use of different buffer systems. Instrumental parameters such as CE capillary position, interface adjustment and contamination problems are also discussed. Separation was performed using uncoated fused silica capillaries with 75 {mu}m i.d. and 70 cm length. The optimum conditions were found to be: Separation voltage 30 kV, positive polarity, capillary temperature 288.15 K and a buffer concentration of 100 mmol l{sup -1} Tricine-NH{sub 3} adjusted to pH 7.2. Sample preparation was performed so as to minimize oxidation and heavy metal contamination of the samples. The high molecular mass protein matrix was reduced by acetonitrile precipitation. For commercial MT preparations the relative standard deviations (R.S.D) in the retention times were 0.9% for MT-1 and 1.9% for MT-2; the R.S.D.'s in the peak areas were less than 6% for MT-1 and 16% for MT-2, respectively. Under optimized conditions the MLPs in the real samples could be separated efficiently in less than 10 min. By comparison with the migration times of commercially available MT preparations, two of the observed peaks could be assigned to MT-1 and MT-2.

  16. Optimization of capillary electrophoresis-inductively coupled plasma mass spectrometry for species analysis of metallothionein-like proteins extracted from liver tissues of Elbe-bream and Roe deer

    International Nuclear Information System (INIS)

    Proefrock, Daniel; Prange, Andreas; Schaumloeffel, Dirk; Ruck, Wolfgang

    2003-01-01

    Species analysis of metallothionein-like proteins (MLP) in liver tissues from Elbe-Bream (Abramis brama L.) and Roe Deer (Capreolus capreolus L.) using capillary electrophoresis (CE) combined with inductively coupled plasma mass spectrometry detection is described. In order to allow systematic development of the method, commercially available metallothionein (MT) preparations of rabbit liver were used. Optimum separation efficiency was obtained by investigating the influence of parameters such as voltage, capillary temperature, buffer concentration, buffer pH and the use of different buffer systems. Instrumental parameters such as CE capillary position, interface adjustment and contamination problems are also discussed. Separation was performed using uncoated fused silica capillaries with 75 μm i.d. and 70 cm length. The optimum conditions were found to be: Separation voltage 30 kV, positive polarity, capillary temperature 288.15 K and a buffer concentration of 100 mmol l -1 Tricine-NH 3 adjusted to pH 7.2. Sample preparation was performed so as to minimize oxidation and heavy metal contamination of the samples. The high molecular mass protein matrix was reduced by acetonitrile precipitation. For commercial MT preparations the relative standard deviations (R.S.D) in the retention times were 0.9% for MT-1 and 1.9% for MT-2; the R.S.D.'s in the peak areas were less than 6% for MT-1 and 16% for MT-2, respectively. Under optimized conditions the MLPs in the real samples could be separated efficiently in less than 10 min. By comparison with the migration times of commercially available MT preparations, two of the observed peaks could be assigned to MT-1 and MT-2

  17. Mutant huntingtin protein expression and blood-spinal cord barrier dysfunction in huntington disease.

    Science.gov (United States)

    Sciacca, Giacomo; Cicchetti, Francesca

    2017-12-01

    The aim of the study was to assess the distribution, frequency, and specific location of mutant huntingtin protein (mHTT) aggregates-the pathological hallmark of Huntington disease (HD)-within the various compartments of the spinal cord and their potential impact on the local vasculature and blood-spinal cord barrier (BSCB). We performed a series of postmortem immunohistochemical and immunofluorescent stainings, as well as Western blot analyses, on cervical and lumbar sections of the spinal cord in patients diagnosed with HD (n = 11 of all grades of disease severity) along with sex- and age-matched healthy controls (n = 9). We observed that mHTT was preferably expressed within the anterior horn of the gray matter, in both cervical and lumbar sections. At the cellular level, mHTT aggregates were more often encountered in the extracellular matrix but could also be observed within cell bodies and neurites as well as within the endothelium of blood vessels with an increase in the density of small blood vessels in cervical sections of HD cases. These vasculature changes were accompanied with features of BSCB leakage, as assessed by the presence of increased levels of fibrinogen in the surrounding parenchyma and enhanced leukocyte infiltration. This alteration in BSCB integrity may be explained, in part, by the dysregulation we found in some of the main proteins associated with it such as junctional adhesion molecule-1 and vascular endothelial cadherin. These observations have important implications for our understanding of HD pathology and may also have significant therapeutic implications. Ann Neurol 2017;82:981-994. © 2017 American Neurological Association.

  18. Binding characteristics of arylamine, o-toluidine, in rodent blood proteins, hemoglobin and albumin

    International Nuclear Information System (INIS)

    DeBord, D.G.; Swearengin, T.F.; Booth-Jones, A.B.; Wissinger, L.A.

    1991-01-01

    o-Toluidine (OT), an arylamine used as an anti-oxidant in the tire and rubber industry, is a suspect human carcinogen. In order to develop a method for biological monitoring of OT, the authors have examined the binding characteristics of OT to the blood proteins, albumin (Alb) and hemoglobin (Hb). Male Sprague-Dawley rats were treated with 10, 20, 40, 50, or 100 mg/kg [ 14 C0]-OT ip and terminated at 2, 4, 8, 18, or 24 hrs. Alb and Hb were isolated from the blood. OT binding was determined by radioactivity associated with Alb and Hb. Maximum OT binding occurred at 4 hrs for Alb and 24 hrs for Hb. The radioactivity associated with Alb was 2x greater than that associated with Hb at 4 hrs, and approximately 30% greater at 24 hrs. Similar OT binding to Alb was observed at the 50 and 100 mg/kg doses, while for Hb maximum binding was seen at 100 mg/kg. Extended time points were studied to determine which blood protein would be more useful for biological monitoring purposes. Rats were treated with 100 mg/kg bw [ 14 C]-OT and terminated at 1, 2 and 4 wks. The remaining radioactivity associated with Alb was 12% after 1 wk, and < 2% after 2 and 4 wks. Conversely, the radioactivity associated with Hb was 64% after 1 wk, 52% by 2 wks, and 24% by 4 wks. These results suggest that Hb may be more useful than Alb as a biological monitoring tool for OT

  19. Denaturing gradient gel electrophoresis

    International Nuclear Information System (INIS)

    Kocherginskaya, S.A.; Cann, I.K.O.; Mackie, R.I.

    2005-01-01

    It is worthwhile considering that only some 30 species make up the bulk of the bacterial population in human faeces at any one time based on the classical cultivation-based approach. The situation in the rumen is similar. Thus, it is practical to focus on specific groups of interest within the complex community. These may be the predominant or the most active species, specific physiological groups or readily identifiable (genetic) clusters of phylogenetically related organisms. Several 16S rDNA fingerprinting techniques can be invaluable for selecting and monitoring sequences or phylogenetic groups of interest and are described below. Over the past few decades, considerable attention was focussed on the identification of pure cultures of microbes on the basis of genetic polymorphisms of DNA encoding rRNA such as ribotyping, amplified fragment length polymorphism and randomly amplified polymorphic DNA. However, many of these methods require prior cultivation and are less suitable for use in analysis of complex mixed populations although important in describing cultivated microbial diversity in molecular terms. Much less attention was given to molecular characterization of complex communities. In particular, research into diversity and community structure over time has been revolutionized by the advent of molecular fingerprinting techniques for complex communities. Denaturing or temperature gradient gel electrophoresis (DGGE/TGGE) methods have been successfully applied to the analysis of human, pig, cattle, dog and rodent intestinal populations

  20. Nitrogen balance, microbial protein synthesis and blood metabolites in fattening of male Bali cattle fed ration with different protein levels in smallholder farms

    Directory of Open Access Journals (Sweden)

    P. K. Tahuk

    2018-03-01

    Full Text Available Research was aimed to determine nitrogen balance, microbial protein synthesis, and blood metabolites of male Bali cattle fattening fed ration with different protein level in smallholder farms North Central Timor, Province of East Timor Tenggara, Indonesia. The cattle used were 18 heads aged 2 to 2.5 years with initial body weight of 229.86±12.46 kg. The cattle were randomly divided into three treatment groups. The T0 group was given feed the same as traditional fattening cattle practices by farmers,T1 group fed ration containing 12% crude protein (CP and 72% total digestible nutrients (TDN, andT2 group fedration containing 15% CP and 72%TDN. Cattle were fed individually for 90 days and drinkingwater ad libitum. The data were analyzedby analysis of variance.Results of research indicated the nitrogen balance, and blood urea nitrogen between T1 and T2 were relatively similar, but those were higher (P<0.05 than T0 . In contrast, microbial proteins synthesis, and blood glucose at 0, 4, and 6 hours before and after feeding were relatively similar between the groups. Blood glucose of T2 at 2 hours after intake were higher (P <0.05 than T0, but was not different with T1 . It can be concluded, that the fattening maleBali cattle fed ration containing 12% CP and 72% TDNimprovedthe nitrogen balance and blood metabolites, butit was no positive effect on the microbial proteins and N synthesis.

  1. Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper.

    Science.gov (United States)

    Jiang, Weidong; Mao, Ying Qing; Huang, Ruochun; Duan, Chaohui; Xi, Yun; Yang, Kai; Huang, Ruo-Pan

    2014-01-31

    Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation

    DEFF Research Database (Denmark)

    Such-Sanmartín, Gerard; Bache, Nicolai; Callesen, Anne K

    2015-01-01

    UNLABELLED: Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further....... BIOLOGICAL SIGNIFICANCE: The interest for MS-based biomarker analysis in body fluids is steadily increasing as proteomics methodology translates into clinical laboratories. We describe a method for detection of 5 distinct proteins of low mass and medium to low abundance, which are of interest in anti...... aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers...

  3. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    International Nuclear Information System (INIS)

    MacDonald, P.N.; Ong, D.E.; Bok, D.

    1990-01-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

  4. Matrix-assisted laser desorption/ionization time of flight mass spectrometry peptide mass fingerprints and post source decay: a tool for the identification and analysis of phloem proteins from Cucurbita maxima Duch. separated by two-dimensional polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Haebel, S; Kehr, J

    2001-08-01

    A combination of gel electrophoresis and mass spectrometry was used to analyze the soluble proteins from phloem sap of Cucurbita maxima Duch. Phloem proteins were separated using two-dimensional gel electrophoresis. Coomassie-stained spots were cut out and subjected to tryptic digestion. To identify proteins, peptide mass fingerprints were determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. In addition, MALDI-TOF post source decay measurements were used to obtain partial sequence information for the proteins. Results from both approaches were used for database searches. In this study, 17 proteins in the mass range 5-50 kDa were analyzed. Of these proteins six could be clearly identified, seven showed significant homologies to known plant proteins, and four were not significantly homologous to database entries. The present study suggests that the applied method is feasible for a large-scale analysis and identification of phloem proteins derived from different organs or from plants kept under various physiological conditions.

  5. Blood levels of glial fibrillary acidic protein (GFAP in patients with neurological diseases.

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    Christoph A Mayer

    Full Text Available BACKGROUND AND PURPOSE: The brain-specific astroglial protein GFAP is a blood biomarker candidate indicative of intracerebral hemorrhage in patients with symptoms suspicious of acute stroke. Comparably little, however, is known about GFAP release in other neurological disorders. In order to identify potential "specificity gaps" of a future GFAP test used to diagnose intracerebral hemorrhage, we measured GFAP in the blood of a large and rather unselected collective of patients with neurological diseases. METHODS: Within a one-year period, we randomly selected in-patients of our university hospital for study inclusion. Patients with ischemic stroke, transient ischemic attack and intracerebral hemorrhage were excluded. Primary endpoint was the ICD-10 coded diagnosis reached at discharge. During hospital stay, blood was collected, and GFAP plasma levels were determined using an advanced prototype immunoassay at Roche Diagnostics. RESULTS: A total of 331 patients were included, covering a broad spectrum of neurological diseases. GFAP levels were low in the vast majority of patients, with 98.5% of cases lying below the cut-off that was previously defined for the differentiation of intracerebral hemorrhage and ischemic stroke. No diagnosis or group of diagnoses was identified that showed consistently increased GFAP values. No association with age and sex was found. CONCLUSION: Most acute and chronic neurological diseases, including typical stroke mimics, are not associated with detectable GFAP levels in the bloodstream. Our findings underline the hypothesis that rapid astroglial destruction as in acute intracerebral hemorrhage is mandatory for GFAP increase. A future GFAP blood test applied to identify patients with intracerebral hemorrhage is likely to have a high specificity.

  6. FORMATION OF LONG-LIVED REACTIVE SPECIES OF BLOOD SERUM PROTEINS BY THE ACTION OF HEAT

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    V. E. Ivanov

    2014-01-01

    Full Text Available Background: Proteins oxidized by the action of X-rays represent long-lived reactive species, which trigger the secondary generation of reactive oxygen species (ROS. A change in the hydrogen peroxide (H2O2 content induced by various physical impacts may be an important factor of the therapeutic effect and the adaptation of the organism to unfavorable environmental conditions. Moderate hyperthermia and a number of physiotherapeutic procedures leading to a local warming of tissues are widely used in medical practice. However, the biological mechanisms of their curative effect are poorly understood. The prolonged generation of H2O2 long-lived reactive protein species (LRPS after heating may be one of the mechanisms of activation of protective cellular mechanisms and thus to contribute to overcoming the disease. Aim: To investigate if the serum proteins bovine serum albumin (BSA and bovine gamma-globulin (BGG can form LRPS under moderate hyperthermia and show that heat induces LRPS, which in turn continuously generate ROS, in particular H2O2. Materials and methods: LRPS were studied by measuring the heat-induced chemiluminescence of protein solutions using a specially elaborated highly sensitive photon-counting chemiluminometer Biotoks-7 AM. The Results: Here we studied the possibility of formation of long-lived species of the blood serum proteins BSA and BGG in air-saturated solutions under the action of heat. It is shown that heat induces the generation of long-lived protein species, which in turn generate ROS (1O2, О2-•, OH•, H2O2. The formation of the long-lived reactive species of BSA and BGG with a half-life of about 4 h induced by moderate hyperthermia was revealed using the chemiluminescence of protein solutions. It was found that long-lived reactive species of BSA and BGG cause prolonged generation of H2O2. Conclusion: Thus, we found a new fundamental property of serum proteins: by the action of moderate heating, they are able to

  7. DNA typing by capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, N.

    1997-10-08

    Capillary electrophoresis is becoming more and more important in nucleic acid analysis including DNA sequencing, typing and disease gene measurements. This work summarized the background of DNA typing. The recent development of capillary electrophoresis was also discussed. The second part of the thesis showed the principle of DNA typing based on using the allelic ladder as the absolute standard ladder in capillary electrophoresis system. Future work will be focused on demonstrating DNA typing on multiplex loci and examples of disease diagnosis in the on-line format of PCR-CE. Also capillary array electrophoresis system should allow high throughput, fast speed DNA typing. Only the introduction and conclusions for this report are available here. A reprint was removed for separate processing.

  8. Oxidative Modification of Blood Serum Proteins in Multiple Sclerosis after Interferon Beta and Melatonin Treatment

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    Monika Adamczyk-Sowa

    2017-01-01

    Full Text Available Multiple sclerosis (MS is a disease involving oxidative stress (OS. This study was aimed at examination of the effect of melatonin supplementation on OS parameters, especially oxidative protein modifications of blood serum proteins, in MS patients. The study included 11 control subjects, 14 de novo diagnosed MS patients with the relapsing-remitting form of MS (RRMS, 36 patients with RRMS receiving interferon beta-1b (250 μg every other day, and 25 RRMS patients receiving interferon beta-1b plus melatonin (5 mg daily. The levels of N′-formylkynurenine, kynurenine, dityrosine, carbonyl groups, advanced glycation products (AGEs, advanced oxidation protein products (AOPP, and malondialdehyde were elevated in nontreated RRSM patients. N′-Formylkynurenine, kynurenine, AGEs, and carbonyl contents were decreased only in the group treated with interferon beta plus melatonin, while dityrosine and AOPP contents were decreased both in the group of patients treated with interferon beta and in the group treated with interferon beta-1b plus melatonin. These results demonstrate that melatonin ameliorates OS in MS patients supporting the view that combined administration of interferon beta-1b and melatonin can be more effective in reducing OS in MS patients than interferon beta-1b alone.

  9. Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

    Science.gov (United States)

    Murphy, Sandra; Dowling, Paul; Ohlendieck, Kay

    2016-01-01

    The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins. PMID:28248237

  10. Clinical value of indicators of cationic proteins, leukocytes myeloperoxidase and fibronectin blood plasma in viral meningitis in children

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    O. G. Kimirilova

    2017-01-01

    Full Text Available Objective: was to establish clinical and diagnostic value of cytochemical indices of peripheral blood leukocytes (cationic protein and myeloperoxidase, fibronectin blood plasma to assess the severity, predict the course and outcome of viral meningitis in children.Subjects and methods. In 450 patients with viral meningitis (enterovirus, arbovirus, parotitic, herpesviral, adenovirus etiology at the age of 14 years, the parameters of the microbicidal system of leukocytes (cation proteins, myeloperoxidase and fibronectin blood plasma were determined. Etiological diagnosis of meningitis was confirmed by release of viral RNA from blood and cerebrospinal fluid by the polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA.The results and conclusion. Found that severe, prolonged duration, lethal outcome of viral meningitis in children are accompanied by sugnificant suppression of cationic proteins, myeloperoxidase, fibronectin blood plasma, maximally expressed in lethal outcomes, compared with the severe form, but with a favorable outcome and control. Settings imbalance cationic proteins, myeloperoxidase, fibronectin blood plasma are objective criteria of the adaptation syndrome that reflects the state of the phagocytosis system in viral meningitis in children and can be considered as additional criteria for predicting the course and outcome of disease.

  11. Spectrophotometric determination of total proteins in blood plasma: a comparative study among dye-binding methods

    Directory of Open Access Journals (Sweden)

    Dimas Augusto Morozin Zaia

    2005-05-01

    Full Text Available A comparative study between the biuret method (standard method for total proteins and spectrophotometric methods using dyes (Bradford, 3',3",5',5"-tetrabromophenolphthalein ethyl ester-TBPEE, and erythrosin-B was carried out for the determination of total proteins in blood plasma from rats. Bradford method showed the highest sensitivity for proteins and biuret method showed the lowest. For all the methods, the absorbance for different proteins (BSA, casein, and egg albumin was measured and Bradford method showed the lowest variation of absorbance. The concentration of total protein obtained by using Bradford method was not statistically different (p>0.05 from concentration of total protein obtained by the biuret method. But in regard to erythrosin-B and TBPEE methods the concentrations of total protein were statistically different (pA determinação de proteínas totais em plasma sangüíneo é importante em diversas áreas de pesquisa. Um estudo comparativo entre o método de biureto (método padrão para proteínas totais e diversos métodos que utilizam corantes (Bradford, tetrabromofenolftaleína etil éster-TBPEE, e eritrosina-B foi realizado para a determinação de proteínas totais em plasma sangüíneo de ratos. O método de Bradford mostrou a maior sensibilidade para proteínas e o de biureto a menor. Para todos os métodos, as absorbâncias para diferentes proteínas (BSA, caseína, e ovoalbumina foram medidas e o método de Bradford mostrou a menor variação da absorbância. Utilizando o método de Bradford a concentração de proteínas totais obtida não foi estatisticamente diferente (p>0.05 daquela obtida pelo método do biureto. Porém, para os métodos da eritrosina-B e TBPEE as concentrações de proteínas totais foram estatisticamente diferentes (p<0.05 da obtida pelo método de biureto. Portanto o método de Bradford pode ser utilizado no lugar do método de biureto para a determinação de proteínas totais em plasma sangüíneo.

  12. Does a lower carbohydrate protein substitute impact on blood phenylalanine control, growth and appetite in children with PKU?

    Science.gov (United States)

    Gokmen-Ozel, Hulya; Ferguson, Carol; Evans, Sharon; Daly, Anne; MacDonald, Anita

    2011-01-01

    In children with phenylketonuria (PKU), it is possible that high carbohydrate protein substitutes may adversely affect blood phenylalanine control. We evaluated if a low carbohydrate, 'ready-to-drink' protein substitute would impact on short term blood phenylalanine control, weight and appetite in children with PKU aged 3-10 years. This was a 3-part, 5-week randomised, controlled, crossover study in which two different carbohydrate/protein-equivalent ratios in protein substitute [control protein substitute (CPS) median 1:1; trial protein substitute (TPS) 0.5:1] were compared. The effects on feeding behaviour, weight change and phenylalanine concentrations were studied. Fourteen children (12 boys; median age 6.3 y, range 3 to 9.7 y) with PKU on diet were recruited from 2 treatment centres. Phenylalanine control did not deteriorate with TPS and remained unchanged between pre-study and CPS (p = 0.783). No statistical differences were noted in energy intake between the two study parts. Any changes in weight were similar between the two groups and there was limited change in feeding behaviour. This study suggests that the carbohydrate/protein-equivalent ratio of protein substitutes can be reduced to 0.5:1 with no loss of blood phenylalanine control or adverse effect on weight gain in children with PKU. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. VanabinP, a novel vanadium-binding protein in the blood plasma of an ascidian, Ascidia sydneiensis samea.

    Science.gov (United States)

    Yoshihara, Masao; Ueki, Tatsuya; Watanabe, Takahiro; Yamaguchi, Nobuo; Kamino, Kei; Michibata, Hitoshi

    2005-09-25

    Some ascidians accumulate high levels of the transition metal vanadium in their blood cells. The process of vanadium accumulation has not yet been elucidated. In this report, we describe the isolation and cDNA cloning of a novel vanadium-binding protein, designated as VanabinP, from the blood plasma of the vanadium-rich ascidian, Ascidia sydneiensis samea. The predicted amino acid sequence of VanabinP was highly conserved and similar to those of other Vanabins. The N-terminus of the mature form of VanabinP was rich in basic amino acid residues. VanabinP cDNA was originally isolated from blood cells, as were the other four Vanabins. However, Western blot analysis revealed that the VanabinP protein was localized to the blood plasma and was not detectable in blood cells. RT-PCR analysis and in situ hybridization indicated that the VanabinP gene was transcribed in some cell types localized to peripheral connective tissues of the alimentary canal, muscle, blood cells, and a portion of the branchial sac. Recombinant VanabinP bound a maximum of 13 vanadium(IV) ions per molecule with a Kd of 2.8 x 10(-5) M. These results suggest that VanabinP is produced in several types of cell, including blood cells, and is immediately secreted into the blood plasma where it functions as a vanadium(IV) carrier.

  14. Estimation of protein concentration at high sensitivity using SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling.

    Science.gov (United States)

    Arrell, Miriam S; Kálmán, Franka

    2016-11-01

    3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS-CGE with LIF detection (SDS-CGE-LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N-termini, which vary in number and accessibility for different proteins. This work investigates the accuracy of estimation of protein concentration with SDS-CGE-LIF in real biological samples, where a different protein must be used as a standard. Sixteen purified proteins varying in molecular weight, structure, and sequence were labeled with FQ at constant mass concentration applying a commonly used procedure for SDS-CGE-LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a RSD of 36%. This compares favorably with other less sensitive methods for estimation of protein concentration such as SDS-CGE-UV and SDS-PAGE-Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the number of labels bound with UHPLC-ESI-QTOF-MS revealed large variations in the labeling efficiency (percentage of labels to the number of labeling sites given by the sequence) for different proteins (from 3 to 30%). This explains the observation that fluorescence per mole of protein was not proportional to the number of lysines in the sequence. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Urinary albumin, protein excretion and circadian blood pressure in patients with fibromyalgia.

    Science.gov (United States)

    Afsar, Baris; Burucu, Rukuye

    2013-09-01

    Recent evidence suggests that patients with fibromyalgia (FM) have increased oxidative stress, inflammation, endothelial dysfunction and autonomic dysfunction. These factors are also shown to be responsible for increased urinary albumin and protein excretion and deranged circadian blood pressure (BP). However, no study has examined the 24-h urinary albumin excretion (UAE), 24-h urinary protein excretion (UPE) and 24-h ambulatory BP measurements in FM patients. The sociodemographic, laboratory parameters, depressive symptoms, sleep problems and 24-h ambulatory BPs were measured for all patients. Diagnosis of FM was based on the criteria for the classification of FM by the American College of Rheumatology. After diagnosis of FM, these patients underwent to complete the Fibromyalgia Impact Questionnaire (FIQ). In total, 30 patients with FM and 61 patients without FM were included. Among FM patients, the average number of tender points was 13.1 ± 1.57 and the mean FIQ score was 57.9 ± 8.86. The number of tender points did not show any correlation with office and ambulatory BPs. There were also no correlations between the number of tender points, UPE and UAE. The stepwise linear regression did not show any relation between UPE and FM. However, 24-h UAE was independently correlated with office systolic BP (P 0.008) and the presence of FM (P 0.045). The logistic regression analysis revealed no association between FM and non-dipping status. We suggest that circadian blood pressure and UPE are not independently associated with FM. However, UAE was related with the presence of FM. Studies are needed to confirm our findings and to highlight pathophysiologic mechanisms.

  16. Investigation of two blood proteins binding to Cantharidin and Norcantharidin by multispectroscopic and chemometrics methods

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Rong; Cheng, Zhengjun, E-mail: ncczj1112@126.com; Li, Tian; Jiang, Xiaohui

    2015-01-15

    The interactions of Cantharidin/Norcantharidin (CTD/NCTD) with two blood proteins, i.e., bovine serum albumin (BSA) and bovine hemoglobin (BHb), have been investigated by the fluorescence, UV–vis absorption, and FT-IR spectra under imitated physiological condition. The binding characteristics between CTD/NCTD and BSA/BHb were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism of two blood proteins with CTD/NCTD is a static quenching. Moreover, the experimental data were further analyzed based on multivariate curve resolution-alternating least squares (MCR-ALS) technique to obtain the concentration profiles and pure spectra for three species (BSA/BHb, CTD/NCTD and CTD/NCTD–BSA/BHb complexes) which existed in the interaction procedure. The number of binding sites n and binding constants K{sub b} were calculated at various temperatures. The thermodynamic parameters (such as, ΔG, ΔH, and ΔS) for BSA–CTD/NCTD and BHb–CTD/NCTD systems were calculated by the Van’t Hoff equation and also discussed. The distance r between CTD/NCTD and BSA/BHb were evaluated according to Förster no-radiation energy transfer theory. The results of Fourier transform infrared (FT-IR), synchronous fluorescence and three-dimensional fluorescence spectra showed that the conformations of BSA/BHb altered with the addition of CTD/NCTD. In addition, the effects of common ions on the binding constants of BSA–CTD/NCTD and BHb–CTD/NCTD systems were also discussed.

  17. Electrophoresis: The Basics (by D. M. Hawcroft)

    Science.gov (United States)

    Voige, William H.

    1999-01-01

    D. M. Hawcroft. Oxford University Press: Oxford, 1997. 142 + xii pp. Index. ISBN 0-19-963563-3. $100.00. This concise monograph is one of a series on techniques in widespread use in biochemistry and cell and molecular biology. It seeks to present, in compact and readable form, the fundamentals of electrophoresis and does so very well. Both theory and practice are included, but emphasis is on the latter. Although the preface makes it clear that this book is intended for biologists, it also deserves a place in a truly complete chemistry library. The book is logically organized. Each of the nine chapters corresponds to either a step in an electrophoresis experiment (e.g., Chapter 7: Visualization of Separated Materials) or a major application (Chapter 4: The Electrophoresis of Native and Denatured Proteins). It is written as though the reader is getting ready to begin doing electrophoresis for the first time and needs a survey of the technique and its applications. A question that occurred to me repeatedly as I read through the book is: Exactly how did the author intend it to be used? One can view the book as either a text or a laboratory manual. As a resource that might be used as a supplementary text in a graduate or upper-division undergraduate course, it does an admirable job of presenting a thorough overview of modern electrophoresis. The figures and diagrams are exceptionally clear and present useful comparisons of results that can be obtained under a variety of conditions (e.g., the resolution of DNA fragments obtained with otherwise identical wedge and normal gels). Not all its explanations, however, are as cogent. It defines how the two portions of a discontinuous gel differ but fails to explain clearly how the porosity and pH differences result in the stacking effect, which is such a gel's primary advantage. Having it on hand as a laboratory manual would be much like having colleagues who are experts in all phases of electrophoresis to consult or to go to

  18. Regulation of Exacerbated Immune Responses in Human Peripheral Blood Cells by Hydrolysed Egg White Proteins

    Science.gov (United States)

    Lozano-Ojalvo, Daniel; Molina, Elena; López-Fandiño, Rosina

    2016-01-01

    The anti-allergic potential of egg white protein hydrolysates (from ovalbumin, lysozyme and ovomucoid) was evaluated as their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs), as well as the release of pro-inflammatory factors and generation of reactive oxygen species from Th1-stimulated peripheral blood leukocytes (PBLs). The binding to IgE of egg allergic patients was determined and the peptides present in the hydrolysates were identified. The hydrolysates with alcalase down-regulated the production of Th2-biased cytokines and the secretion of IgE to the culture media of Th2-skewed PBMCs, and they significantly neutralized oxidative stress in PBLs. The hydrolysates of ovalbumin and ovomucoid with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. The hydrolysates with alcalase, in addition to equilibrating Th2 differentiation, exhibited a low IgE-binding. Therefore, they would elicit mild allergic reactions while retaining T cell-stimulating abilities, which might correlate with an anti-allergic benefit. PMID:27007699

  19. Regulation of Exacerbated Immune Responses in Human Peripheral Blood Cells by Hydrolysed Egg White Proteins.

    Directory of Open Access Journals (Sweden)

    Daniel Lozano-Ojalvo

    Full Text Available The anti-allergic potential of egg white protein hydrolysates (from ovalbumin, lysozyme and ovomucoid was evaluated as their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs, as well as the release of pro-inflammatory factors and generation of reactive oxygen species from Th1-stimulated peripheral blood leukocytes (PBLs. The binding to IgE of egg allergic patients was determined and the peptides present in the hydrolysates were identified. The hydrolysates with alcalase down-regulated the production of Th2-biased cytokines and the secretion of IgE to the culture media of Th2-skewed PBMCs, and they significantly neutralized oxidative stress in PBLs. The hydrolysates of ovalbumin and ovomucoid with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. The hydrolysates with alcalase, in addition to equilibrating Th2 differentiation, exhibited a low IgE-binding. Therefore, they would elicit mild allergic reactions while retaining T cell-stimulating abilities, which might correlate with an anti-allergic benefit.

  20. Blood Pyrrole-Protein Adducts--A Biomarker of Pyrrolizidine Alkaloid-Induced Liver Injury in Humans.

    Science.gov (United States)

    Ruan, Jianqing; Gao, Hong; Li, Na; Xue, Junyi; Chen, Jie; Ke, Changqiang; Ye, Yang; Fu, Peter Pi-Cheng; Zheng, Jiang; Wang, Jiyao; Lin, Ge

    2015-01-01

    Pyrrolizidine alkaloids (PAs) induce liver injury (PA-ILI) and is very likely to contribute significantly to drug-induced liver injury (DILI). In this study we used a newly developed ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-MS)-based method to detect and quantitate blood pyrrole-protein adducts in DILI patients. Among the 46 suspected DILI patients, 15 were identified as PA-ILI by the identification of PA-containing herbs exposed. Blood pyrrole-protein adducts were detected in all PA-ILI patients (100%). These results confirm that PA-ILI is one of the major causes of DILI and that blood pyrrole-protein adducts quantitated by the newly developed UHPLC-MS method can serve as a specific biomarker of PA-ILI.

  1. Simulation of Two Dimensional Electrophoresis and Tandem Mass Spectrometry for Teaching Proteomics

    Science.gov (United States)

    Fisher, Amanda; Sekera, Emily; Payne, Jill; Craig, Paul

    2012-01-01

    In proteomics, complex mixtures of proteins are separated (usually by chromatography or electrophoresis) and identified by mass spectrometry. We have created 2DE Tandem MS, a computer program designed for use in the biochemistry, proteomics, or bioinformatics classroom. It contains two simulations--2D electrophoresis and tandem mass spectrometry.…

  2. Identification of ectoproteins of human platelets. Combination of radioactive labelling and two-dimensional electrophoresis

    NARCIS (Netherlands)

    Sixma, J.J.; Schiphorst, M.E.

    1980-01-01

    Two-dimensional gel electrophoresis combining isoelectric focussing of reduced or non-reduced proteins in the first dimension with electrophoresis in sodium dodecyl sulfate polyacrylamide gels in the second dimension enabled us to identify 25 ectoproteins in the non-reduced state and 32 in the

  3. Proteomic analysis and sperm physiopathology: the two dimensional difference in gel electrophoresis approach

    OpenAIRE

    Vazquez, Monica Hebe

    2015-01-01

    Two-dimensional (2D) gel electrophoresis coupled with protein identification using mass spectrometry (MS) is a procedure extensively used to obtain a catalog of several thousand proteins in a single gel. These evaluations provide information that complements and extends the results retrieved from messenger RNA (mRNA) profiling using microarray technologies (1). Basically, the first step on the 2D gel electrophoresis analysis is based on protein separation according to [...]. Fil: Vazquez, ...

  4. CHARACTERIZATION OF ENU-INDUCED MUTATIONS IN RED BLOOD CELL STRUCTURAL PROTEINS

    Directory of Open Access Journals (Sweden)

    Katrina Kildey

    2013-03-01

    Full Text Available Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b demonstrated increased RBC count, haemoglobin (Hb and haematocrit (HCT. The third Spnb1 mutation (spectrin-1β c and mutation in Epb4.1 (band 4.1 did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of

  5. Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

    Science.gov (United States)

    Arts, Remco; den Hartog, Ilona; Zijlema, Stefan E; Thijssen, Vito; van der Beelen, Stan H E; Merkx, Maarten

    2016-04-19

    Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

  6. Extracorporeal irradiation of calves blood. Effects on: the lymphocytes, the blood-platelet function, seric proteins, and fibrinogen

    International Nuclear Information System (INIS)

    Hollard, D.; Suscillon, M.; Benabid, Y.; Concord, E.; Ivanoff, M.; Laurent, M.; Rambaud, F.

    1969-01-01

    The present paper reports the results obtained after extracorporeal irradiation of circulating blood of calves. Animals are divided in 3 groups as follows: - control animals: blood circulation without irradiation; - calves which received 40000 rads during 24 hours of continuous irradiation; - calves which received the same dose, during a period of 5 days (5 hours every day). The more interesting results are: - the early lymphopenia which persists for 7 or 8 weeks and may be in relationship with the change of immunoglobulins; - a constant hyperfibrinemia (12 g/l) never reported, as far as we know, by authors using I.E.C. Several hypothesis are advanced to explain this phenomenon. (authors) [fr

  7. Capillary electrophoresis-based assessment of nanobody affinity and purity

    NARCIS (Netherlands)

    Haselberg, Rob; Oliveira, Sabrina; van der Meel, Roy; Somsen, Govert W; de Jong, Gerhardus J

    2014-01-01

    Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a

  8. Blood profile of proteins and steroid hormones predicts weight change after weight loss with interactions of dietary protein level and glycemic index.

    Directory of Open Access Journals (Sweden)

    Ping Wang

    2011-02-01

    Full Text Available Weight regain after weight loss is common. In the Diogenes dietary intervention study, high protein and low glycemic index (GI diet improved weight maintenance.To identify blood predictors for weight change after weight loss following the dietary intervention within the Diogenes study.Blood samples were collected at baseline and after 8-week low caloric diet-induced weight loss from 48 women who continued to lose weight and 48 women who regained weight during subsequent 6-month dietary intervention period with 4 diets varying in protein and GI levels. Thirty-one proteins and 3 steroid hormones were measured.Angiotensin I converting enzyme (ACE was the most important predictor. Its greater reduction during the 8-week weight loss was related to continued weight loss during the subsequent 6 months, identified by both Logistic Regression and Random Forests analyses. The prediction power of ACE was influenced by immunoproteins, particularly fibrinogen. Leptin, luteinizing hormone and some immunoproteins showed interactions with dietary protein level, while interleukin 8 showed interaction with GI level on the prediction of weight maintenance. A predictor panel of 15 variables enabled an optimal classification by Random Forests with an error rate of 24±1%. A logistic regression model with independent variables from 9 blood analytes had a prediction accuracy of 92%.A selected panel of blood proteins/steroids can predict the weight change after weight loss. ACE may play an important role in weight maintenance. The interactions of blood factors with dietary components are important for personalized dietary advice after weight loss.ClinicalTrials.gov NCT00390637.

  9. Plasma protein concentrations of the young and adult Amazona brasiliensis parrots

    Directory of Open Access Journals (Sweden)

    Schmidt Elizabeth Moreira Dos Santos

    2017-01-01

    Full Text Available Introduction. The Red-tailed Amazon parrot (Amazona brasiliensis is an endangered species of the Psittacine family, and for which various data are important for a comprehensive preservation plan. Data about plasma protein gel electrophoresis of Amazon parrot blood are scarce. The purpose of this study was to determine plasma protein concentrations and concentrations of major protein bands in blood of young and adult Red-tailed Amazon parrot (Amazona brasiliensis. Materials and Methods. Blood samples from eight young and eight adult healthy free-living parrots were obtained. Plasma protein concentration and fractions were determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE. Mann-Whitney U test was used to compare variables. Results and Conclusions. Six major protein bands with the following molecular weights were identified by SDS-PAGE: 170 kDa, 117 kDa, 85 kDa (putative ovotransferrin, 60 kDa, 45 kDa and 23 kDa. Adult parrots had significantly higher concentrations of total proteins, albumin and other proteins with similar mobility (around 60 kDa. Young birds had significantly higher levels of 23kDa proteins. The concentration of putative ovotransferrin (85 kDa was not different between young and adult parrots. Plasma protein gel electrophoresis patterns in Red-tailed Amazon parrots are similar between young and adult animals, but specific protein bands differ in their absolute concentrations. This finding should be taken into consideration when clinical pathology data are analysed.

  10. Profil Protein Trypanosoma evansi dari Daerah Geografis Berbeda di Indonesia Tahun 2012-2014 dengan Sodium Dodecil Sulphate Polyacrylamide Gel Electrophoresis (TRYPANOSOMA EVANSI PROTEIN PROFILE OF DIFFERENT GEOGRAPHICAL AREAS ORIGIN IN INDONESIA

    Directory of Open Access Journals (Sweden)

    Fitrine Ekawasti

    2018-01-01

    Full Text Available Surra outbreak in 2012 has led to more than 1,700 animals have died in the province of East Nusa Tenggara (NTT Indonesia. Surra case sporadically continues throughout the year in various areas, especially Kalimantan, Banten as well as other areas. Some reports reveal differences in protein profiles among multiple isolates of T. evansi. Therefore the purpose of this research were to find out the protein profile of each isolate T. evansi in Indonesia and the possible biological differences among them. Eleven isolates originating from the province of East Nusa Tenggara, South Kalimantan and Central Kalimantan, Banten, Lampung and Bengkulu has been isolated and purified Using DEAE. Trypanosoma isolate were frezeethawing repeatedly to obtain soluble protein. Furthermore, soluble protein is treated with heating or without heating and then each was run on SDS PAGE with Coomassie Blue staining. The protein profiles of all isolates were compared each other. The results showed that eleven isolates of T. evansi in Indonesia has a very diverse protein profile. Then for the purposes of development of diagnostic kit can be used whole lysate cell (WCL as stock antigen in serological test process.

  11. Concentration of total proteins in blood plasma of chickens hatched from irradiated eggs with low dose gamma radiation

    International Nuclear Information System (INIS)

    Vilic, M.; Kraljevic, P.; Miljanic, S.; Simpraga, M.

    2005-01-01

    It is known that low-dose ionising radiation may have stimulating effects on chickens. Low doses may also cause changes in the concentration of blood plasma total proteins, glucose and cholesterol in chickens. This study investigates the effects of low dose gamma-radiation on the concentration of total proteins in the blood plasma of chickens hatched from eggs irradiated with a dose of 0.15 Gy on incubation days 7 and 19. Results were compared with the control group (chickens hatched from non-irradiated eggs). After hatching, all other conditions were the same for both groups. Blood samples were drawn from the heart, and later from the wing vein on days 1, 3, 5, 7,10, 20, 30 and 42. The concentration of total proteins was determined spectrophotometrically using Boehringer Mannheim GmbH optimised kits. The concentration of total proteins in blood plasma in chickens hatched from eggs irradiated with 0.15 Gy on incubation day 7 showed a statistically significant decrease on the sampling day 3 (P less than 0.05) and 7 (P less than 0.01). The concentration of total proteins in blood plasma in chickens hatched from eggs irradiated with 0.15 Gy on incubation day 19 showed a statistically significant increase only on sampling day 1 (P less than 0.05). These results suggest that exposure of eggs to 0.15 Gy of gamma-radiation on the 7th and 19th day of incubation could produce different effects on the protein metabolism in chickens.(author)

  12. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hao-Tsai Cheng

    2016-01-01

    Full Text Available Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established. Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE profiling quality, including number of protein spots and spot distribution. Results. The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin. Conclusions. Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images.

  13. The IgE-dependent pathway in allergic transfusion reactions: involvement of donor blood allergens other than plasma proteins.

    Science.gov (United States)

    Matsuyama, Nobuki; Yasui, Kazuta; Amakishi, Etsuko; Hayashi, Tomoya; Kuroishi, Ayumu; Ishii, Hiroyuki; Matsukura, Harumichi; Tani, Yoshihiko; Furuta, Rika A; Hirayama, Fumiya

    2015-07-01

    On transfusion, several plasma proteins can cause anaphylaxis in patients deficient in the corresponding plasma proteins. However, little is known about other allergens, which are encountered much more infrequently. Although it has been speculated that an allergen-independent pathway underlying allergic transfusion reactions (ATRs) is elicited by biological response modifiers accumulated in blood components during storage, the exact mechanisms remain unresolved. Furthermore, it is difficult even to determine whether ATRs are induced via allergen-dependent or allergen-independent pathways. To distinguish these two pathways in ATR cases, we established a basophil activation test, in which the basophil-activating ability of supernatants of residual transfused blood of ATR cases to whole blood basophils was assessed in the presence or absence of dasatinib, an inhibitor of IgE-mediated basophil activation. Three of 37 supernatants from the platelet concentrates with ATRs activated panel blood basophils in the absence, but not in the presence, of dasatinib. The basophil activation was inhibited by treatment of anti-fish collagen I MoAb in one case, suggesting that the involvement of fish allergens may have been present in donor plasma. We concluded that unknown non-plasma proteins, some of which had epitopes similar to fish antigens, in blood component may be involved in ATRs via an allergen/IgE-dependent pathway.

  14. Determination of olanzapine in whole blood using simple protein precipitation and liquid chromatography-tandem mass spectrometry

    DEFF Research Database (Denmark)

    Nielsen, Marie Katrine Klose; Johansen, Sys Stybe

    2009-01-01

    A simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of the antipsychotic drug olanzapine in whole blood using dibenzepine as internal standard (IS). After acidic methanol-induced protein precipitation...

  15. Film Thickness Dependence of Protein Adsorption from Blood Serum and Plasma onto Poly(sulfobetaine)-Grafted Surfaces

    Czech Academy of Sciences Publication Activity Database

    Vaisocherová, Hana; Yang, W.; Chen, S.; Cheng, G.; Xue, H.; Zhang, J.; Jiang, S.; Li, W.

    2008-01-01

    Roč. 24, č. 17 (2008), s. 9211-9214 ISSN 0743-7463 Institutional research plan: CEZ:AV0Z20670512 Keywords : zwitterionic poly(sulfobetaine) * protein adsorption * blood serum and plasma Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.097, year: 2008

  16. The effect of varying protein levels on blood chemistry, food consumption, and behavior of captive seaducks

    Science.gov (United States)

    Wells-Berlin, A. M.; Perry, M.C.; Olsen, Glenn H.

    2005-01-01

    The Chesapeake Bay is a primary wintering area for scoters and the long-tailed ducks (Clangia hyemalis) that migrate along the Atlantic Flyway. Recently, the Chesapeake Bay had undergone an ecosystem shift and little is known about how this is affecting the seaduck populations. We are determining what are the preferred food sources of the seaducks wintering on the Bay and analyzing the factors influencing prey selection whether it is prey composition, energy assimilated, prey availability, or a combination of any or all of these factors. We have established a captive colony of surf (Melanitta perspicillata) and white-winged scoters (Melanitta fusca) as well as long-tailed ducks at Patuxent Wildlife Research Center to allow us to examine these factors in a more controlled environment. This project contains a multitude of experiments and the resultant data will be compiled into a compartmental model on the feeding ecology of seaducks wintering on the Bay. The first experiment entailed feeding groups of each species (four ducks per pen of equal sex ratio, if possible, and four pens per species) three diets varying in percent protein levels from November to February. Each diet was randomly assigned to each pen and the amount of food consumed was recorded each day. New feed was given when all existing food was consumed. Behavioral trials and blood profiles were completed on all study birds to determine the effects of the varying diets. There were no significant differences in food consumption, blood chemistry, and behavior detected at the 5% level among the diets for all three species of interest. There was a seasonal effect determined based on the food consumption data for white-winged scoters, but not for surf scoters or long-tailed ducks. The blood profiles of the surf scoters were compared to blood profiles of wild surf scoters and a there was no difference detected at the 5% level. As a health check of the ducks an aspergillosis test was run on the blood obtained

  17. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

    National Research Council Canada - National Science Library

    Dovichi, Norman J

    2005-01-01

    .... Capillary sieving electrophoresis and capillary micellar electrophoresis were used to characterize proteins in single cells in one-dimensional separations, while the two techniques were combined...

  18. Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein

    Directory of Open Access Journals (Sweden)

    Malin R. Reiten

    2018-01-01

    Full Text Available The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs derived from non-transgenic goats that lack PrPC (PRNPTer/Ter compared with cells from normal (PRNP+/+ goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS, revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

  19. Influence of puerperium on the serum proteins of Saanen goats, obtained by the analysis of electrophoresis on sodium dodecyl sulphate-polyacrylamide gel / Influência do puerpério sobre o proteinograma sérico de caprinos da raça Saanen obtido por eletroforese em gel de poliacrilamida

    Directory of Open Access Journals (Sweden)

    Eduardo Harry Birgel Junior

    2009-10-01

    Full Text Available Aiming to evaluate the puerperal influence on the proteinogram of Saanen goats, 108 samples of blood serum from 12 goats were collected, and the results were presented at nine times: just after parturition, 1, 3, 5, 7, 10, 15, 21 and 30 days after parturition. Total amount of serum proteins were determined by the biuret technique, and the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE was used to the protein fractionation. In this last method, 17 protein bands were observed, from which molecular weights varied between 25 KDa and 275 KDa. In addition, it was possible to identify the following protein fractions: immunoglobulin A (180 KDa, ceruloplasmin (115 KDa, transferrin (79 KDa, albumin (65 KDa, heavy-chain immunoglobulin G (58 KDa, haptoglobin (45 KDa, acid glycoprotein (37 KDa and light-chain immunoglobulin G (28 KDa. Another 9 nonidentified protein fractions presented, each molecular weights equal to 275 KDa, 140 KDa, 125 KDa, 103 KDa, 95 KDa, 41 KDa, 35 KDa, 30 Kda and 25 KDa. The results allow us to conclude that by the first week of puerperium, an improvement of acid glycoprotein occurs, whereas those others protein fractions do not suffer any puerperal influence.Com o objetivo de avaliar a influência do puerpério no proteinograma de caprinos da raça Saanen foram colhidas 108 amostras de soro sangüíneo de 12 cabras, sendo os resultados apresentados em nove momentos: imediatamente após a parição, 1, 3, 5, 7, 10, 15, 21 e 30 dias após o parto. Os teores séricos de proteína total foram determinados pela técnica do biureto, e para o fracionamento das proteínas foi realizada a eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE. No método utilizado foram observadas 17 bandas protéicas, cujos pesos moleculares (PM variaram de 25 KDa a 275 KDa, sendo possível a identificação das seguintes frações protéicas: imunoglobulina A (180 KDa, ceruloplasmina (115 KDa, transferrina

  20. C-reactive protein and chitinase 3-like protein 1 as biomarkers of spatial redistribution of retinal blood vessels on digital retinal photography in patients with diabetic retinopathy.

    Science.gov (United States)

    Cekić, Sonja; Cvetković, Tatjana; Jovanović, Ivan; Jovanović, Predrag; Pesić, Milica; Stanković Babić, Gordana; Milenković, Svetislav; Risimić, Dijana

    2014-08-20

    The aim of the study was to investigate the correlation between the levels of C-reactive protein (CRP) and chitinase 3-like protein 1 (YKL-40) in blood samples with morpohometric parameters of retinal blood vessels in patients with diabetic retinopathy. Blood laboratory examination of 90 patients included the measurement of glycemia, HbA1C, total cholesterol, LDL-C, HDL-C, triglycerides and CRP. Levels of YKL-40 were detected and measured in serum by ELISA (Micro VueYKL-40 EIA Kit, Quidel Corporation, San Diego, USA). YKL-40 correlated positively with diameter and negatively with number of retinal blood vessels. The average number of the blood vessels per retinal zone was significantly higher in the group of patients with mild non-proliferative diabetic retinopathy than in the group with severe form in the optic disc and all five retinal zones. The average outer diameter of the evaluated retinal zones and optic disc vessels was significantly higher in the group with severe compared to the group with mild diabetic retinopathy. Morphological analysis of the retinal vessels on digital fundus photography and correlation with YKL-40 may be valuable for the follow-up of diabetic retinopathy.

  1. Changes in protein abundance between tender and tough meat from bovine Longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis

    DEFF Research Database (Denmark)

    Bjarnadóttir, S G; Hollung, K; Høy, M

    2012-01-01

    The aim of this study was to find potential biomarkers for meat tenderness in bovine Longissimus thoracis muscle and to compare results from isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis (2-DE) analysis. The experiment included 4 tender and 4...

  2. Effects of protein and vitamin B deficiency on blood parameters and myenteric neurons of the colon of rats

    Directory of Open Access Journals (Sweden)

    Sant'Ana Débora de Mello Gonçales

    2001-01-01

    Full Text Available The aims of this work were to evaluate the effects of the deficient ingestion of protein and vitamin B on the biochemical and hematologic parameters and on the NADH- and NADPH-diaphorase positive myenteric neurons. The control animals (n=10 received commercial chow and the experimental rats (n=10 received chow with protein level reduced to 8% during 120 days. At the time of killing blood was collected for assessment of the blood and hematologic parameters and the ascending colon for quantitative analysis of the neurons of the myenteric plexus. It was observed that the reduction of the protein level to 8% coupled to the reduction of the levels of vitamin B in adult rats neither led to qualitative or quantitative changes on red or white blood cells, nor decreased globulin levels, induced the formation of edema or gave rise to clinical signs typical of protein or vitamin B deficiency. On the other hand, the experimental protocol led to less weight gain, change on the body composition with fat deposition; decrease of the values of serum total protein and albumin; reduction of the area of colon and density of nitrergic and NADH-diaphorase myenteric neurons inferior to the expected.

  3. Differential outcomes of Zika virus infection in Aedes aegypti orally challenged with infectious blood meals and infectious protein meals.

    Science.gov (United States)

    Huang, Yan-Jang S; Lyons, Amy C; Hsu, Wei-Wen; Park, So Lee; Higgs, Stephen; Vanlandingham, Dana L

    2017-01-01

    infection in mosquitoes. Infectious whole blood meals and infectious bovine serum albumin meals containing ZIKV were orally presented to two different groups of Ae. aegypti through membrane feeding. At 7 and 14 days post infection, infectious viruses were detected and viral dissemination from gut to other mosquito tissues was analyzed in orally challenged mosquitoes with 50% tissue culture infectious dose method on Vero76 cells. Zika virus infection was significantly impaired among mosquitoes orally challenged with infectious protein meals as compared to infectious whole blood meals. These results indicate the importance of the blood meal in the infection process of arboviruses in mosquitoes. It provides the basis for future studies to identify critical components in the blood of vertebrate hosts that facilitate arbovirus infection in mosquitoes.

  4. Effects of different concentrations of Maytenus ilicifolia (Espinheira Santa) on labelling of red blood cells and blood proteins with Technetium-99m

    International Nuclear Information System (INIS)

    Oliveira, Joelma F.; Braga, Ana Cristina S.; Bezerra, Roberto Jose A.C.; Bernardo-Filho, Mario

    1999-01-01

    The use of natural products in all over the world has been increased in Brazil as well as in other countries. Maytenus ilicifolia is commonly used in popular medicine. The labeling of red blood cells (RBC) with technetium-99m ( 99m Tc) have been for many studies in nuclear medicine. This labeling procedure depends on a reducing agent and stannous chloride is normally used. Here, we investigate if the extract of Maytenus ilicifolia is capable to alter the labeling of RBC and blood proteins with 99m Tc. Blood samples were incubated with Maytenus ilicifolia. Stannous chloride solution and Tc-99m were. Blood was centrifuged and plasma (P) and blood cells (C) were isolated. Samples of P or C were precipitated with trichloroacetic acid, centrifuged and IF and IF were separated. The percentage of radioactivity (% ATI) in C, IF-P and IF-C was calculated. The %ATI in decreased in C from 93.6±2.3 to 29.0±2.7, on FI-P from 77.6±1.2 to 7.5 ±1.0 and on FI-C from 80.0±3.4 to 12.6±4.8. Once in RBC labeling procedure with 99m Tc depends on the presence of stannous (+2), the substances of the natural product could increase the valence of stannous (+2) to stannic (+4). This fact would decrease the labeling of blood elements with 99m Tc. (author)

  5. Effects of previous protein intake on rectal temperature, blood glucose, plasma thyroid hormone and minerals by laying hens during a forced molt

    International Nuclear Information System (INIS)

    Rodrigues, G.A.; Moraes, V.M.B.; Cherici, I; Furlan, R.L.; Macari, M.

    1991-01-01

    The effects of forced molting on blood glucose, rectal temperature, plasma T4, T3 and minerals were studied in hens previously fed rations with different protein contents (14, 17 and 20% crude protein). Blood samples were obtained from brachial veins for blood glucose, T4 and T3 were measured by radioimmunoassay, and plasma minerals were determined by atomic absorption spectroscopy. Blood glucose and rectal temperature were reduced during fasting regardless of previous protein intake. Pre molting T4 plasma level was higher in laying hens fed higher protein ration, but feed deprivation reduced T 4 and T 3 concentrations irrespective of protein intake, except T 4 level for 14% crude protein fed birds that increased during fasting. The data obtained in this experiment suggest that previous protein intake does not interfere with the metabolic changes during forced molt. (author). 19 refs, 1 fig, 4 tabs

  6. Gestational dietary protein is associated with sex specific decrease in blood flow, fetal heart growth and post-natal blood pressure of progeny.

    Science.gov (United States)

    Hernandez-Medrano, Juan H; Copping, Katrina J; Hoare, Andrew; Wapanaar, Wendela; Grivell, Rosalie; Kuchel, Tim; Miguel-Pacheco, Giuliana; McMillen, I Caroline; Rodgers, Raymond J; Perry, Viv E A

    2015-01-01

    The incidence of adverse pregnancy outcomes is higher in pregnancies where the fetus is male. Sex specific differences in feto-placental perfusion indices identified by Doppler assessment have recently been associated with placental insufficiency and fetal growth restriction. This study aims to investigate sex specific differences in placental perfusion and to correlate these changes with fetal growth. It represents the largest comprehensive study under field conditions of uterine hemodynamics in a monotocous species, with a similar long gestation period to the human. Primiparous 14 mo heifers in Australia (n=360) and UK (n=180) were either individually or group fed, respectively, diets with differing protein content (18, 14, 10 or 7% crude protein (CP)) from 60 d prior to 98 days post conception (dpc). Fetuses and placentae were excised at 98 dpc (n = 48). Fetal development an median uterine artery blood flow were assessed monthly from 36 dpc until term using B-mode and Doppler ultrasonography. MUA blood flow to the male feto-placental unit increased in early pregnancy associated with increased fetal growth. Protein restriction before and shortly after conception (-60 d up to 23 dpc) increased MUA diameter and indices of velocity during late pregnancy, reduced fetal heart weight in the female fetus and increased heart rate at birth, but decreased systolic blood pressure at six months of age. Sex specific differences both in feto-placental Doppler perfusion indices and response of these indices to dietary perturbations were observed. Further, maternal diet affected development of fetal cardiovascular system associated with altered fetal haemodynamics in utero, with such effects having a sex bias. The results from this study provide further insight into the gender specific circulatory differences present in the fetal period and developing cardiovascular system.

  7. Gestational dietary protein is associated with sex specific decrease in blood flow, fetal heart growth and post-natal blood pressure of progeny.

    Directory of Open Access Journals (Sweden)

    Juan H Hernandez-Medrano

    Full Text Available The incidence of adverse pregnancy outcomes is higher in pregnancies where the fetus is male. Sex specific differences in feto-placental perfusion indices identified by Doppler assessment have recently been associated with placental insufficiency and fetal growth restriction. This study aims to investigate sex specific differences in placental perfusion and to correlate these changes with fetal growth. It represents the largest comprehensive study under field conditions of uterine hemodynamics in a monotocous species, with a similar long gestation period to the human. Primiparous 14 mo heifers in Australia (n=360 and UK (n=180 were either individually or group fed, respectively, diets with differing protein content (18, 14, 10 or 7% crude protein (CP from 60 d prior to 98 days post conception (dpc. Fetuses and placentae were excised at 98 dpc (n = 48. Fetal development an median uterine artery blood flow were assessed monthly from 36 dpc until term using B-mode and Doppler ultrasonography. MUA blood flow to the male feto-placental unit increased in early pregnancy associated with increased fetal growth. Protein restriction before and shortly after conception (-60 d up to 23 dpc increased MUA diameter and indices of velocity during late pregnancy, reduced fetal heart weight in the female fetus and increased heart rate at birth, but decreased systolic blood pressure at six months of age.Sex specific differences both in feto-placental Doppler perfusion indices and response of these indices to dietary perturbations were observed. Further, maternal diet affected development of fetal cardiovascular system associated with altered fetal haemodynamics in utero, with such effects having a sex bias. The results from this study provide further insight into the gender specific circulatory differences present in the fetal period and developing cardiovascular system.

  8. Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

    Energy Technology Data Exchange (ETDEWEB)

    Nie, Song [Biological Sciences Division; Shi, Tujin [Biological Sciences Division; Fillmore, Thomas L. [Biological Sciences Division; Schepmoes, Athena A. [Biological Sciences Division; Brewer, Heather [Biological Sciences Division; Gao, Yuqian [Biological Sciences Division; Song, Ehwang [Biological Sciences Division; Wang, Hui [Biological Sciences Division; Rodland, Karin D. [Biological Sciences Division; Qian, Wei-Jun [Biological Sciences Division; Smith, Richard D. [Biological Sciences Division; Liu, Tao [Biological Sciences Division

    2017-08-11

    Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined with precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.

  9. Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

    Science.gov (United States)

    Hazzalin, Catherine A; Mahadevan, Louis C

    2017-01-01

    Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

  10. Determination of olanzapine in whole blood using simple protein precipitation and liquid chromatography-tandem mass spectrometry

    DEFF Research Database (Denmark)

    Nielsen, Marie Katrine Klose; Johansen, Sys Stybe

    2009-01-01

    A simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of the antipsychotic drug olanzapine in whole blood using dibenzepine as internal standard (IS). After acidic methanol-induced protein precipitation......, and stability. The absolute recovery obtained was 103% for olanzapine and 68% for IS. An LOQ of 0.005 mg/kg olanzapine in whole blood was achieved. Inter- and intraday precision were less than 11% within concentrations from 0.01 to 0.50 mg/kg, and the accuracy ranged from 85 to 115%. The method was subsequently...

  11. Expanding the capabilities of microfluidic gradient elution moving boundary electrophoresis for complex samples.

    Science.gov (United States)

    Strychalski, Elizabeth A; Henry, Alyssa C; Ross, David

    2011-08-15

    Gradient elution moving boundary electrophoresis (GEMBE) is a robust, continuous injection separation technique that uses electrophoresis to drive electrically charged analytes into a capillary or microfluidic channel for detection, while opposing electroosmosis and controlled variable pressure-driven flow prevent other sample components-for example, cells, proteins, or particulates in complex samples that can interfere with analysis-from entering the channel. This work expands the sample-in/answer-out analytical capabilities of GEMBE for complex samples by demonstrating the quantitative analysis of anions, implementing aqueous background electrolyte (BGE) solutions at neutral pH, and introducing the use of additives to the sample solution to optimize performance. Dirt was analyzed quantitatively, with the sole preparatory step of suspension in an aqueous BGE solution at neutral pH, for dissolved chloride, nitrite, nitrate, sulfate, and oxalate using GEMBE with capacitively-coupled contactless conductivity detection. In addition to altering the pH of the BGE solution, optimization of the analysis of dirt and whole blood was achieved using various commercially available additives. These results, taken together with previous demonstrations of GEMBE for the analysis of complex samples, underscore the uncomplicated versatility of GEMBE, facilitate effective analysis of biological complex samples using BGE solutions at physiological pH, and offer a sufficient set of techniques and tools to build a foundation for the analysis of a broad range of complex samples.

  12. Associations of sodium, potassium and protein intake with blood pressure and hypertension in Switzerland.

    Science.gov (United States)

    Glatz, Nicolas; Chappuis, Aline; Conen, David; Erne, Paul; Péchère-Bertschi, Antoinette; Guessous, Idris; Forni, Valentina; Gabutti, Luca; Muggli, Franco; Gallino, Augusto; Hayoz, Daniel; Binet, Isabelle; Suter, Paolo; Paccaud, Fred; Bochud, Murielle; Burnier, Michel

    2017-03-21

    Nutritional factors play an important role in the regulation of blood pressure and in the development of hypertension. In this analysis, we explored the associations of 24-hour urinary Na+, K+ and urea excretion with blood pressure levels and the risk of hypertension in the Swiss population, taking regional linguistic differences into account. The Swiss Survey on Salt is a population based cross-sectional study that included 1336 subjects from the three main linguistic regions (French, German and Italian) of Switzerland. Blood pressure was measured with a validated oscillometric Omron HEM 907 device. Hypertension was defined as current antihypertensive treatment or a mean systolic blood pressure >140 mm Hg and/or diastolic >90 mm Hg, based on eight blood pressure measurements performed at two visits. Na+, K+ and urea excretion were assessed in 24-hour urine collections. We use multiple logistic/linear regressions to explore the associations of urine Na+, K+ and urea with blood pressure / hypertension, taking into account potential confounders and effect modifiers. The prevalence of hypertension was 30%, 26% and 17% in the German-, French- and Italian- speaking regions respectively, (p-value across regions blood pressure and hypertension. Urinary K+ excretion tended to be negatively associated with blood pressure but this was not significant (p = 0.08). Hypertensive people had a higher 24-hour urinary Na+/K+ ratio than normotensive people (p = 0.003). Urinary urea excretion was associated with neither blood pressure nor hypertension. Participants from the German-speaking region had a higher likelihood of having a high systolic blood pressure. We confirm a high prevalence of elevated blood pressure in Swiss adults, including regional differences. In Switzerland, urinary Na+ excretion is associated positively with blood pressure and hypertension, independently of urinary K+ and urea excretion. The observed differences in blood pressure levels across linguistic regions

  13. Blood flow restriction prevents muscle damage but not protein synthesis signaling following eccentric contractions

    Science.gov (United States)

    Sudo, Mizuki; Ando, Soichi; Poole, David C; Kano, Yutaka

    2015-01-01

    There is a growing body of evidence to suggest that resistance training exercise combined with blood flow restriction (BFR) increases muscle size and strength in humans. Eccentric contraction (ECC) frequently induces severe muscle damage. However, it is not known whether and to what extent muscle damage occurs following ECC + BFR due to the difficulty of conducting definitive invasive studies. The purpose of this study was to examine muscle fiber damage following ECC + BFR at the cellular level. High-intensity ECC was purposefully selected to maximize the opportunity for muscle damage and hypertrophic signaling in our novel in vivo animal model. Male Wistar rats were assigned randomly to the following groups: ECC and ECC + BFR at varying levels of occlusion pressure (140, 160, and 200 Torr). In all conditions, electrical stimulation was applied to the dorsiflexor muscles simultaneously with electromotor-induced plantar flexion. We observed severe histochemical muscle fiber damage (area of damaged fibers/total fiber area analyzed) following ECC (26.4 ± 4.0%). Surprisingly, however, muscle damage was negligible following ECC + BFR140 (2.6 ± 1.2%), ECC+BFR160 (3.0 ± 0.5%), and ECC + BFR200 (0.2 ± 0.1%). Ribosomal S6 kinase 1 (S6K1) phosphorylation, a downstream target of rapamycin (mTOR)-phosphorylation kinase, increased following ECC + BFR200 as well as ECC. In contrast, S6K1 phosphorylation was not altered by BFR alone. The present findings suggest that ECC combined with BFR, even at high exercise intensities, may enhance muscle protein synthesis without appreciable muscle fiber damage. PMID:26149281

  14. Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    2011-03-01

    Full Text Available Botulinum neurotoxin (BoNT potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP to link biotinylated molecules to glycophorin A (GPA on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

  15. Subcompartmentalisation of proteins in the rhoptries correlates with ordered events of erythrocyte invasion by the blood stage malaria parasite.

    Directory of Open Access Journals (Sweden)

    Elizabeth S Zuccala

    Full Text Available Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction - the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion.

  16. Ratcheted electrophoresis of Brownian particles

    Energy Technology Data Exchange (ETDEWEB)

    Kowalik, Mikołaj; Bishop, Kyle J. M., E-mail: kjmbishop@engr.psu.edu [Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802 (United States)

    2016-05-16

    The realization of nanoscale machines requires efficient methods by which to rectify unbiased perturbations to perform useful functions in the presence of significant thermal noise. The performance of such Brownian motors often depends sensitively on their operating conditions—in particular, on the relative rates of diffusive and deterministic motions. In this letter, we present a type of Brownian motor that uses contact charge electrophoresis of a colloidal particle within a ratcheted channel to achieve directed transport or perform useful work against an applied load. We analyze the stochastic dynamics of this model ratchet to show that it functions under any operating condition—even in the limit of strong thermal noise and in contrast to existing ratchets. The theoretical results presented here suggest that ratcheted electrophoresis could provide a basis for electrochemically powered, nanoscale machines capable of transport and actuation of nanoscale components.

  17. Ajuste de metodologia da eletroforese de proteínas para obtenção de fenótipos isoenzimáticos de moranga Adjustment of the methodology of protein electrophoresis to obtain isozymatic phenotypes of pumpkin

    Directory of Open Access Journals (Sweden)

    Antonio Teixeira do Amaral Júnior

    1994-01-01

    Full Text Available Modificações no procedimento eletroforético foram introduzidas para uma satisfatória obtenção de fenótipos isoenzimáticos de moranga (Cucurbita maxima Duchesne. Extratos de folhas definitivas de oito acessos de moranga foram submetidos à análise eletroforética para revelação dos seguintes sistemas: esterase (EST; fosfatase ácida (ACP; transaminase do glutamato oxaloacetato (GOT, desidrogenase do malato (MDH e peroxidase (PO. Os resultados apontaram a ineficiência do tampão extrator utilizado e satisfatória qualidade do gel de amido de milho nas resoluções dos sistemas EST, ACP, GOT e MDH. Para o sistema PO, adequado efeito de peneiramento molecular só foi possível com a utilização do amido hidrolisado de batata (Sigma. Ajustes no tempo de cozimento do gel, manuseio da corrente elétrica, preparo de soluções corantes e tempo de revelação indicaram os melhores protocolos ao pro-cedimento eletroforético.Changes in the electrophoresis procedures were introduced to obtain a better isozymatic phenotypes of pumpkin (Cucurbita maxima Duchesne. Extracts of eight access of pumpkin's leaves were subjected to electrophoresis analysis in order to reveal the following systems: esterase (EST, acid phosphatase (ACP, glutamate oxaloacetate transaminase (GOT, malato dehydrogenase (MDH and peroxidase (PO. The results pointed out the inefficiency of the extractor buffer used, and the satisfactory quality of maize starch gel electrophoresis in the resolution of EST, ACP, GOT and MDH systems. For PO system a better effect of "molecular sieving" was only possible when potato starch gel (Sigma was used. Adjusting the starch cooking time, handling the electric current, preparing the staining solutions and revealing time indicated the best protocol to the electrophoresis process.

  18. DNA Sequencing by Capillary Electrophoresis

    Science.gov (United States)

    Karger, Barry L.; Guttman, Andras

    2009-01-01

    Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA sequencing methods have evolved from the labor intensive slab gel electrophoresis, through automated multicapillary electrophoresis systems using fluorophore labeling with multispectral imaging, to the “next generation” technologies of cyclic array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes was only possible by the advent of modern sequencing technologies that was a result of step by step advances with a contribution of academics, medical personnel and instrument companies. While next generation sequencing is moving ahead at break-neck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of capillary electrophoresis in DNA sequencing based in part of several of our articles in this journal. PMID:19517496

  19. Marked increase in rat red blood cell membrane protein glycosylation by one-month treatment with a cafeteria diet

    Directory of Open Access Journals (Sweden)

    Laia Oliva

    2015-07-01

    Full Text Available Background and Objectives. Glucose, an aldose, spontaneously reacts with protein amino acids yielding glycosylated proteins. The compounds may reorganize to produce advanced glycosylation products, which regulatory importance is increasingly being recognized. Protein glycosylation is produced without the direct intervention of enzymes and results in the loss of function. Glycosylated plasma albumin, and glycosylated haemoglobin are currently used as index of mean plasma glucose levels, since higher glucose availability results in higher glycosylation rates. In this study we intended to detect the early changes in blood protein glycosylation elicited by an obesogenic diet.Experimental Design. Since albumin is in constant direct contact with plasma glucose, as are the red blood cell (RBC membranes, we analyzed their degree or glycosylation in female and male rats, either fed a standard diet or subjected to a hyper-energetic self-selected cafeteria diet for 30 days. This model produces a small increase in basal glycaemia and a significant increase in body fat, leaving the animals in the initial stages of development of metabolic syndrome. We also measured the degree of glycosylation of hemoglobin, and the concentration of glucose in contact with this protein, that within the RBC. Glycosylation was measured by colorimetric estimation of the hydroxymethylfurfural liberated from glycosyl residues by incubation with oxalate.Results. Plasma glucose was higher in cafeteria diet and in male rats, both independent effects. However, there were no significant differences induced by sex or diet in either hemoglobin or plasma proteins. Purified RBC membranes showed a marked effect of diet: higher glycosylation in cafeteria rats, which was more marked in females (not in controls. In any case, the number of glycosyl residues per molecule were higher in hemoglobin than in plasma proteins (after correction for molecular weight. The detected levels of glucose in

  20. C-reactive protein and white blood cell count do not improve clinical decision-making in acute appendicitis

    DEFF Research Database (Denmark)

    Tind, Sofie; Lassen, Annmarie Touborg; Zimmermann-Nielsen, Erik

    2015-01-01

    INTRODUCTION: Acute appendicitis (AA) remains a diagnostic challenge as indicated by the high rate of unnecessary surgery. Blood samples, primarily C-reactive protein (CRP) and leucocyte counts, are used as a diagnostic supplement despite their relatively low sensitivities and specificities....... However, their influence on diagnostic decision-making has not previously been investigated. The aim of the present study was to investigate if the results of CRP and leucocytes had any positive or negative influence on the decision-making of surgeons handling patients with suspected AA. METHODS...... appendicitis on histology. The surgeons changed their diagnosis in nine cases after assessing blood samples. The changes in the proportion of correct diagnoses, sensitivity, specificity and predictive values after assessing blood samples were not significant. CONCLUSIONS: The results of CRP and leucocyte...

  1. Blood-brain barrier transport and protein binding of flumazenil and iomazenil in the rat: implications for neuroreceptor studies

    DEFF Research Database (Denmark)

    Videbaek, C; Ott, P; Paulson, O B

    1999-01-01

    of blood-brain barrier permeability for two benzodiazepine antagonists were performed in 44 rats by the double-indicator technique. Cerebral blood flow was measured by intracarotid Xe-injection. The apparent permeability-surface product (PSapp) was measured while CBF or bolus composition was changed......The calculated fraction of receptor ligands available for blood-brain barrier passage in vivo (f(avail)) may differ from in vitro (f(eq)) measurements. This study evaluates the protein-ligand interaction for iomazenil and flumazenil in rats by comparing f(eq) and f(avail). Repeated measurements......(avail) and f(eq) as well as the effect of CBF on PSapp can be caused by capillary heterogeneity....

  2. Determination of olanzapine in whole blood using simple protein precipitation and liquid chromatography-tandem mass spectrometry

    DEFF Research Database (Denmark)

    Nielsen, Marie Katrine Klose; Johansen, Sys Stybe

    2009-01-01

    A simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of the antipsychotic drug olanzapine in whole blood using dibenzepine as internal standard (IS). After acidic methanol-induced protein precipitation...... of the whole blood samples, olanzapine and IS were chromatographed on a reversed-phase Zorbax Extend-C(18)-column at pH 9.0. Quantification was performed on a triple-quadrupole mass spectrometer employing electrospray ionization technique operating in multiple reaction monitoring and positive ion mode. Total...... chromatographic run time was 15 min, and calibration curve was linear over the concentration range of 0.005 to 0.50 mg/kg olanzapine in whole blood. The method was validated for selectivity, matrix interference, recovery, linearity, limit of detection, limit of quantitation (LOQ), accuracy, precision...

  3. Serum, plasma, and dried blood spot high sensitivity C-reactive protein enzyme immunoassay for population research

    OpenAIRE

    Brindle, Eleanor; Fujita, Masako; Shofer, Jane; O’Connor, Kathleen A.

    2010-01-01

    C-reactive protein (CRP) is used as a biomarker of morbidity and mortality risk in studies of population health, and is essential to interpretation of several micronutrient biomarkers. There is thus need for a robust high sensitivity CRP (hsCRP) measurement method for large-scale, non-clinical studies. We developed an efficient, inexpensive assay suitable for quantifying CRP across the physiological range using any blood specimen type. The ELISA uses readily available monoclonal antibodies to...

  4. Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

    Science.gov (United States)

    Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

    2013-09-15

    Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Human lymphocyte polymorphisms detected by quantitative two-dimensional electrophoresis

    International Nuclear Information System (INIS)

    Goldman, D.; Merril, C.R.

    1983-01-01

    A survey of 186 soluble lymphocyte proteins for genetic polymorphism was carried out utilizing two-dimensional electrophoresis of 14 C-labeled phytohemagglutinin (PHA)-stimulated human lymphocyte proteins. Nineteen of these proteins exhibited positional variation consistent with independent genetic polymorphism in a primary sample of 28 individuals. Each of these polymorphisms was characterized by quantitative gene-dosage dependence insofar as the heterozygous phenotype expressed approximately 50% of each allelic gene product as was seen in homozygotes. Patterns observed were also identical in monozygotic twins, replicate samples, and replicate gels. The three expected phenotypes (two homozygotes and a heterozygote) were observed in each of 10 of these polymorphisms while the remaining nine had one of the homozygous classes absent. The presence of the three phenotypes, the demonstration of gene-dosage dependence, and our own and previous pedigree analysis of certain of these polymorphisms supports the genetic basis of these variants. Based on this data, the frequency of polymorphic loci for man is: P . 19/186 . .102, and the average heterozygosity is .024. This estimate is approximately 1/3 to 1/2 the rate of polymorphism previously estimated for man in other studies using one-dimensional electrophoresis of isozyme loci. The newly described polymorphisms and others which should be detectable in larger protein surveys with two-dimensional electrophoresis hold promise as genetic markers of the human genome for use in gene mapping and pedigree analyses

  6. Evaluation of some selected blood parameters and histopathology of liver and kidney of rats fed protein-substituted mucuna flour and derived protein rich product.

    Science.gov (United States)

    Ngatchic, Josiane Therese Metsagang; Sokeng, Selestion Dongmo; Njintang, Nicolas Yanou; Maoundombaye, Theophile; Oben, Julius; Mbofung, Carl Moses F

    2013-07-01

    This comparative study reports the nutritional and toxicological characteristics of Mucuna pruriens flour and a protein-rich product developed from it. The protein-rich mucuna product (PRMP) was obtained by the three steps procedure: protein solubilization, heat-coagulation and sieving. Three weeks rats (n=6 per group) were fed for 28 days on standard protein-substituted rat feed with mucuna flour or PRMP. The experimental design was a factorial design with three mucuna accessions (Velvet, Black and White) and two treatments (flour and PRMP). The protein content ranged 27.2-31.5 g/100 g for flour and 58.8-61.1% for PRMP. Processing flour into PRMP led to a significant (p<0.05) reduction of tannins (50%), total polyphenols (50%) and trypsin inhibitors (94%). The rats fed PMRP diets witnessed weight gain similar to casein, while those fed mucuna flour lost weight. The levels of total cholesterol, HDL-cholesterol and LDL-cholesterol observed in animals groups fed mucuna flour and PRMP were significantly lower (p<0.05) than the control group. In addition lymphocytes, granulocytes, red blood cells, hemoglobin and hematocrit of rats fed mucuna flour were significantly (p<0.05) lower than values in other rats groups. Kidneys glomerular sclerosis and high creatinine levels were observed in group fed mucuna flour. PRMP then represents a good alternative of using mucuna proteins for human nutrition. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  7. Effect of a high-protein diet on maintenance of blood pressure levels achieved after initial weight loss

    DEFF Research Database (Denmark)

    Engberink, M F; Geleijnse, J M; Bakker, S J L

    2015-01-01

    Randomized trials have shown significant blood pressure (BP) reductions after increased protein compared with carbohydrate intake, but the effect on BP maintenance after initial weight loss is unclear. We examined the effect of a high-protein diet on the maintenance of reduced BP after weight loss...... in 420 overweight adults from the Diet, Obesity and Genes study. After an 8-week weight-loss period (>8% BW), subjects (42±6 years) were randomized to either a high-protein diet (23-28 en% protein) or a lower-protein control diet (10-15 en% protein) for 26 weeks. BMI after weight loss was 30.3±4.3 kg m......-protein control group. In 191 (pre)hypertensive subjects (baseline systolic BP⩾120 mm Hg), a larger difference was observed (-4.2 mm Hg (-7.7, -0.7), P=0.02). The effect was attenuated after adjustment for initial BP (-3.4 mm Hg (-6.9, -0.03), P=0.048), and after additional adjustment for weight change (-2.7 mm...

  8. Usage of capillary electrophoresis for common hemoglobinopathies screening

    Directory of Open Access Journals (Sweden)

    Alireza Ebrahimi

    2016-06-01

    Full Text Available Hemoglobinopathies are most common inherited disorders in the world; approximately 7 percent of the worldwide population and 5-6 percent of population of Iran are carriers. The hemoglobin disorders inherit as autosomal recessive and are very common in the Mediterranean area and much of the Asia and Africa. The control of this inherited disorders need to genetic counseling and accurate screening by more advanced and more accurate methods. This study explains features of current Iran hemoglobin disorders, nominates the accessible methods for screening them and introduces the capillary zone electrophoresis as a rapid and more accurate method. The required data were extracted of various articles and then for good explanation, current Iran hemoglobinopathies properties were showed in the tables and electropherograms of important hemoglobin disorders in Iran population were provided for help to interpretation results of blood tests by capillary zone electrophoresis method. Hemoglobin disorders are including thalassemias and hemoglobin variants; Disruption in the production and malfunction of globin chains cause types of hemoglobin disorders. We cannot introduce one of clinical laboratory tests as critical and basic method for screening and distinguishing types of inherited hemoglobin disorders as alone. For distinguishing the types of them must be prepared enough information and data of the hemoglobin disorders and for more accurate analysis must be used simultaneously different methods as gel electrophoresis, high performance liquid chromatography, isoelectric focusing, capillary zone electrophoresis or molecular tests. The capillary electrophoresis is an accurate and rapid method for screening types of the hemoglobin disorders. Other side this method cannot analyze all of them, so must be used biochemical, biophysical and molecular methods for confirmation the results. This review showed we can use the capillary electrophoresis and HPLC as two

  9. Usage of Capillary Electrophoresis for screening common Hemoglobinopathies

    Directory of Open Access Journals (Sweden)

    2016-06-01

    Full Text Available Hemoglobinopathies are most common inherited disorders in the world approximately 7 percent of the worldwide population and 5-6 percent of population of Iran are carriers. For control of this inherited hemoglobin disorders need to accurate screening by more advanced and more accurate methods. This study explains features of current Iran hemoglobin disorders, nominates the accessible methods for screening them and introduces the capillary zone electrophoresis as a rapid & more accurate method. The required data were extracted of various articles and then for good explanation, current Iran hemoglobinopathies properties were showed in the tables and electropherograms of important hemoglobin disorders in Iran population were provided for help to interpretation results of blood tests by capillary zone electrophoresis method. Hemoglobin disorders are including thalassemias & hemoglobin variants Disruption in the production and malfunction of globin chains cause types of hemoglobin disorders. We cannot introduce one of clinical laboratory tests as critical and basic method for screening and distinguishing types of inherited hemoglobin disorders as alone. For distinguishing the types of them must be prepared enough information and data of the hemoglobin disorders and for more accurate analysis must be used simultaneously different methods as Gel electrophoresis, High performance liquid chromatography, Isoelectric focusing, Capillary zone electrophoresis or molecular tests. The capillary electrophoresis is an accurate and rapid method for screening types of the hemoglobin disorders. Other side this method cannot analyze all of them, so must be used biochemical, biophysical and molecular methods for confirmation the results. This review showed we can use the capillary electrophoresis and HPLC as two complementary methods for hemoglobinopathies screening. We can analyze by the methods more hemoglobin disorders and decrease more laboratory errors. Moreover

  10. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  11. Effect of an extract of Artemisia vulgaris L. (Mugwort) on the in vitro labeling of red blood cells and plasma proteins with technetium-99m

    International Nuclear Information System (INIS)

    Terra, Danielle Amorim; Brandao-Neto, Jose; Medeiros, Aldo da Cunha; Amorim, Lucia de Fatima; Catanho, Maria Tereza Jansen de Almeida; Fonseca, Adenilson de Souza da; Santos-Filho, Sebastiao David; Bernardo-Filho, Mario

    2007-01-01

    The aim of this work was to evaluate the effect of an extract of the Artemisia vulgaris L. (mugwort) on the labeling of blood constituents with technetium-99m (99mTc). Blood samples from Wistar rats were incubated with a mugwort extract and the radiolabeling of blood constituents was carried out. Plasma and blood cells were separated by centrifugation. Aliquots of plasma and blood cells were also precipitated with trichloroacetic acid and centrifuged to isolate soluble and insoluble fractions of plasma and blood cells. Radioactivity in each fraction was counted and the percentages of radioactivity (%ATI) was calculated. Mugwort extract decreased significantly (p<0.05) the %ATI on the blood compartments and on the blood cells proteins (insoluble fraction). The analysis of the results indicates that the ex