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Sample records for denaturing polymer electrophoresis

  1. 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

  2. Acetic acid denaturing pulsed field capillary electrophoresis for RNA separation.

    Science.gov (United States)

    Li, Zhenqing; Dou, Xiaoming; Ni, Yi; Sumitomo, Keiko; Yamaguchi, Yoshinori

    2010-10-01

    Based on our previous work of in-capillary denaturing polymer electrophoresis, we present a study of RNA molecular separation up to 6.0 kilo nucleotide by pulsed field CE. This is the first systematic investigation of electrophoresis of a larger molecular mass RNA in linear hydroxyethylcellulose (HEC) under pulsed field conditions. The parameters that may influence the separation performance, e.g. gel polymer concentration, modulation depth and pulse frequency, are analyzed in terms of resolution and mobility. For denaturing and separating RNA in the capillary simultaneously, 2 M acetic acid was added into the HEC polymer to serve as separation buffer. Result shows that (i) in pulsed field conditions, RNA separation can be achieved in a wide range of concentration of HEC polymer, and RNA fragments between 0.3 and 0.6 kilo nucleotide are sensitive to the polymer concentration; (ii) under certain pulsed field conditions, RNA fragments move linearly as the modulation depth increases; (iii) 12.5 Hz is the resonance frequency for RNA reorientation time and applied frequency.

  3. Evaluation of denaturing gradient gel electrophoresis (DGGE) used ...

    African Journals Online (AJOL)

    Denaturing gradient gel electrophoresis (DGGE) is a powerful method used to study structure of bacterial communities, without cultivation, based on the diversity of the genes coding for ribosomal RNA. However, the results are strongly dependent on the respective target region of the used primer systems. Therefore, three ...

  4. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique.

    OpenAIRE

    Noll, W W; Collins, M

    1987-01-01

    Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. We have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and th...

  5. Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis

    NARCIS (Netherlands)

    Wu, Y; Hayes, VM; Osinga, J; Mulder, IM; Looman, MWG; Buys, CHCM; Hofstra, RMW

    1998-01-01

    Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest

  6. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique

    International Nuclear Information System (INIS)

    Noll, W.W.; Collins, M.

    1987-01-01

    Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. The authors have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and the reaction mixture is electrophoresed on a denaturing gradient gel. Only the genomic DNA probe hybrids migrate into the gel. Differences in hybrid mobility on the gel indicate base pair changes in the genomic DNA. They have used this technique to identify two polymorphic sites within a 1.2-kilobase region of human chromosome 20. This approach should greatly facilitate the identification of DNA polymorphisms useful for gene linkage studies and the diagnosis of genetic diseases

  7. Conducting polymer electrodes for gel electrophoresis.

    Directory of Open Access Journals (Sweden)

    Katarina Bengtsson

    Full Text Available In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that π-conjugated polymers such as poly(3,4-ethylenedioxythiophene (PEDOT can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation.

  8. Conducting polymer electrodes for gel electrophoresis.

    Science.gov (United States)

    Bengtsson, Katarina; Nilsson, Sara; Robinson, Nathaniel D

    2014-01-01

    In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that π-conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis) systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation.

  9. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

    Science.gov (United States)

    Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

    2010-03-01

    Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

  10. Conducting Polymer Electrodes for Gel Electrophoresis

    OpenAIRE

    Bengtsson, Katarina; Nilsson, Sara; Robinson, Nathaniel D

    2014-01-01

    In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that p-conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis) systems. In this report, we extend our previous result to gel ...

  11. Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

    International Nuclear Information System (INIS)

    Bjoerheim, Jens; Abrahamsen, Torveig Weum; Kristensen, Annette Torgunrud; Gaudernack, Gustav; Ekstroem, Per O.

    2003-01-01

    Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification

  12. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I

    1999-01-01

    to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

  13. Investigating the fermentation of cocoa by correlating denaturing gradient gel electrophoresis profiles and near infrared spectra

    DEFF Research Database (Denmark)

    Nielsen, Dennis Sandris; Snitkjær, Pia; van der Berg, Franciscus Winfried J

    2008-01-01

    demonstrating the microbial succession taking place during the fermentation. Subsequently the DGGE spectra were correlated to the NIR spectra using Partial Least Squares regression models (PLS2). Correlations of 0.87 (bacterial derived DGGE spectra) and 0.81 (yeast derived DGGE spectra) were obtained indicating......Raw cocoa has an astringent, unpleasant taste and flavour, and has to be fermented, dried and roasted in order to obtain the characteristic cocoa flavour and taste. During the fermentation microbial activity outside the cocoa beans induces biochemical and physical changes inside the beans...... of the beans and the chemical processes inside the beans have been carried out previously. Recently it has been shown that Denaturing Gradient Gel Electrophoresis (DGGE) offers an efficient tool for monitoring the microbiological changes taking place during the fermentation of cocoa. Near Infrared (NIR...

  14. New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

    NARCIS (Netherlands)

    Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

    2000-01-01

    A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

  15. On the mobility of partially denatured DNA in gel electrophoresis: a theoretical investigation

    Science.gov (United States)

    Sean, David

    There are technologies which exploit a rapid reduction of the gel electrophoretic mobility of DNA arising from partial denaturation. The underlying phenomenon behind these experiments---the mechanisms which reduce the mobility---are not very well understood. Such is the purpose of my thesis. The first chapter provides a brief introduction to the field of polymer physics. The subjects covered are carefully chosen to directly relate to the forthcoming research. There is a published semi-empirical formula used to model the rapid decrease of mobility which is largely considered to be consistent with experimental data. The second chapter of this thesis demonstrates that there is a fundamental confusion in the literature regarding the fitting parameter Lr, in the said formula. By going back to the original derivation, a physical interpretation can be given to L r. This interpretation yields theoretical values which are consistent with what has been published. However, we find that an underlying assumption---that the effect of the denaturation does not depend on its position along the DNA fragment---may systematically overestimate experimental observations of Lr. To measure the impact of this assumption, a simulation model of DNA is presented. The article presented in the third chapter reveals that indeed, the position of the denatured region affects the migration of the DNA fragment. A refined version of the formula which takes these factors into account is proposed. The simulations also reveal that, for certain fields, an unexpected conformation completely dominates during migration of the fragment. This surprising result: a squid-like conformation, is explored in chapter four.

  16. Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota.

    Science.gov (United States)

    Hoyles, Lesley; Clear, Jessica A; McCartney, Anne L

    2013-08-01

    With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intensity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Comparison of non-electrophoresis grade with electrophoresis grade BIS in NIPAM polymer gel preparation

    Science.gov (United States)

    Khodadadi, Roghayeh; Khajeali, Azim; Farajollahi, Ali Reza; Hajalioghli, Parisa; Raeisi, Noorallah

    2015-01-01

    Introduction:The main objective of this study was to investigate the possibility of replacing electrophoresis cross-linker with non-electrophoresis N, N′-methylenebisacrylamide (BIS) in N-isopropyl acrylamide (NIPAM) polymer gel and its possible effect on dose response. Methods: NIPAM polymer gel was prepared from non-electrophoresis grade BIS and the relaxation rate (R2) was measured by MR imaging after exposing the gel to gamma radiation from Co-60 source. To compare the response of this gel with the one that contains electrophoresis grade BIS, two sets of NIPAM gel were prepared using electrophoresis and non-electrophoresis BIS and irradiated to different gamma doses. Results: It was found that the dose–response of NIPAM gel made from the non-electrophoresis grade BIS is coincident with that of electrophoresis grade BIS. Conclusion:Taken all, it can be concluded that the non-electrophoresis grade BIS not only is a suitable alternative for the electrophoresis grade BIS but also reduces the cost of gel due to its lower price. PMID:26457250

  18. Polymerase chain reaction-based denaturing gradient gel electrophoresis in the evaluation of oral microbiota.

    Science.gov (United States)

    Li, Y; Saxena, D; Barnes, V M; Trivedi, H M; Ge, Y; Xu, T

    2006-10-01

    Clinical evaluation of oral microbial reduction after a standard prophylactic treatment has traditionally been based on bacterial cultivation methods. However, not all microbes in saliva or dental plaque can be cultivated. Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) is a cultivation-independent molecular fingerprinting technique that allows the assessment of the predominant bacterial species present in the oral cavity. This study sought to evaluate the oral microbial changes that occurred after a standard prophylactic treatment with a conventional oral care product using PCR-DGGE. Twelve healthy adults participated in the study. Pooled plaque samples were collected at baseline, 24 h after prophylaxis (T1), and 4 days after toothbrushing with fluoride toothpaste (T4). The total microbial genomic DNA of the plaque was isolated. PCR was performed with a set of universal bacterial 16S rDNA primers. The PCR-amplified 16S rDNA fragments were separated by DGGE. The effects of the treatment and of dental brushing were assessed by comparing the PCR-DGGE fingerprinting profiles. The mean numbers of detected PCR amplicons were 22.3 +/- 6.1 for the baseline group, 13.0 +/- 3.1 for the T1 group, and 13.5 +/- 4.3 for the T4 group; the differences among the three groups were statistically significant (P < 0.01). The study also found a significant difference in the mean similarities of microbial profiles between the baseline and the treatment groups (P < 0.001). PCR-based DGGE has been shown to be an excellent means of rapidly and accurately assessing oral microbial changes in this clinical study.

  19. An improved method for detecting genetic variation in DNA using denaturing gradient gel electrophoresis

    International Nuclear Information System (INIS)

    Takahashi, Norio; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

    1990-05-01

    We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that use of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than use of DNA:DNA heteroduplexes, because preparation of RNA probes is easier than that of DNA probes. Three different 32 P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of a mismatch(es) was detected as a difference in the mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and three thalassemic human β-globin genes. The results from experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human β-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was undertaken in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for detecting heritable variation and should be a powerful tool for detecting fresh mutations in DNA, which occur outside the known restriction sites. (author)

  20. Detection by denaturing gradient gel electrophoresis of ammonia-oxidizing bacteria in microcosms of crude oil-contaminated mangrove sediments.

    Science.gov (United States)

    dos Santos, A C F; Marques, E L S; Gross, E; Souza, S S; Dias, J C T; Brendel, M; Rezende, R P

    2012-01-27

    Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.

  1. Denaturing gradient gel electrophoresis profiling of bacterial communities composition in Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Singh, S.K.; Ramaiah, N.

    of Environmental Biology circleshadowdwnMay 2011circleshadowdwn Introduction The bacteria play a major role in carbon dynamics of marine ecosystems and, the importance of heterotrophic bacteria in marine ecosystem functioning is very well recognized (Azam et al..., 2008). Denaturing gradient gel-electrophoressis (DGGE) based fingerprinting helps estimate the numbers of dominant phylotype in a given sample (Muyzer et al., 1993). Very diverse bacterial assemblages such as those in the soils present many bands...

  2. The detection of Mycoplasma (formerly Eperythrozoon) wenyonii by 16S rDNA PCR and denaturing gradient gel electrophoresis.

    Science.gov (United States)

    McAuliffe, Laura; Lawes, Joanna; Bell, Suzanna; Barlow, Alex; Ayling, Roger; Nicholas, Robin

    2006-10-31

    Although the role of Mycoplasma wenyonii in disease is still subject to some debate, infections have been reported to result in parasitaemia, anaemia, scrotal and hind limb oedema, tachycardia, pyrexia, infertility, swollen teats, prefemoral lymphadenopathy and decreased milk production. Previously, diagnosis of M. wenyonii has been based on blood smears but is not specific for M. wenyonii and can be difficult to interpret. We have previously described the use of PCR and denaturing gradient gel electrophoresis (DGGE) for the detection and differentiation of Mycoplasma species. DGGE enables the rapid and specific identification of Mycoplasma species and is ideally suited to detecting both mixed infections and new and unusual species. In this study, we have used DGGE with universal primers to detect M. wenyonii DNA from blood samples. DGGE can be used on blood samples as a rapid and specific test for M. wenyonii and can also be used as a screening test for other blood borne pathogens.

  3. Changes in the diversity of pig ileal lactobacilli around weaning determined by means of 16S rRNA gene amplification and denaturing gradient gel electrophoresis

    NARCIS (Netherlands)

    Janzcyk, P.; Pieper, R.; Smidt, H.; Souffrant, W.B.

    2007-01-01

    Our study aimed to provide a comprehensive characterization of changes in porcine intestinal Lactobacillus populations around the time of weaning based on 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE). DNA was extracted from the ileal contents of piglets at weaning

  4. Development and application of a selective pcr-denaturing gradient gel electrophoresis approach to detect a recently cultivated Bacillus group predominant in soil

    NARCIS (Netherlands)

    Tzeneva, V.A.; Li, Y.; Felske, A.; Vos, de W.M.; Akkermans, A.D.L.; Vaughan, E.E.; Smidt, H.

    2004-01-01

    The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance

  5. Potential use of gradient denaturing gel electrophoresis in obtaining mutational spectra from human cells

    International Nuclear Information System (INIS)

    Thilly, W.G.

    1985-01-01

    A method is described to isolate mutations in DNA in human cells. When a double-stranded DNA migrates through an electric field on an electrophoretic gel, it is compact hydrodynamic structure relative to the same material in a melted form. Normally the solution in electrophoretic gels is uniform, but a way has been devised to set up a stable gradient of increasing solute concentration in the direction of DNA motion. Thus, as a double-stranded DNA molecule is drawn by the electric field into higher and higher concentrations of urea/formamide, it will eventually reach a point at which the concentration is high enough to melt the lower-melting-point region. The melting results in an essentially immobile structure within the gel so that the position at which the DNA molecule stops on the gradient gel is determined by its melting point, which is uniquely determined by its nucleotide sequence. A single base pair substitution within a low melting point sequence of some 100 base pairs changed the expected melting point by 0.4 0 C and resulted in about a 2-cm displacement under appropriate denaturing gel conditions. This expectation leads to the idea that if a mixture of DNA sequences derived from point mutations within the same restriction fragment were permitted to anneal with a complementary wild-type sequence, the melting point of each type of heteroduplex would differ depending on the kind and position of each mutation

  6. Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko (Radiation Effects Research Foundation, Hiroshima (Japan))

    1993-01-01

    In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE - a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies. 46 refs., 3 figs., 1 tab.

  7. Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

    Science.gov (United States)

    Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M

    2012-08-01

    Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

  8. Bacteria community study of combined periodontal-endodontic lesions using denaturing gradient gel electrophoresis and sequencing analysis.

    Science.gov (United States)

    Li, Hong; Guan, Rui; Sun, Jinghua; Hou, Benxiang

    2014-10-01

    The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared. Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs. Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia. The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.

  9. Bacterial analysis of combined periodontal-endodontic lesions by polymerase chain reaction-denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Xia, Minghui; Qi, Qingguo

    2013-01-01

    We used denaturing gradient gel electrophoresis (DGGE) to compare bacterial profiles in periodontium and root canals of teeth with combined periodontal-endodontic lesions. Samples of dental plaque and necrotic pulp were collected from thirteen extracted teeth with advanced periodontitis. Genomic DNA was extracted for polymerase chain reaction (PCR) analysis using universal bacterial primers. The PCR products were then loaded onto DGGE gels to obtain fractionated bands. Characteristic DGGE bands were excised and DNA was cloned and sequenced. The number of bands, which indicates the number of bacterial species, was compared between dental plaques and necrotic pulp tissues from the same tooth. Although the difference was statistically significant (P bacteria species were present in both the periodontal pockets and root canals of the same tooth; however, periodontal bacteria did not always invade the root canals, and some bacteria in root canals were not present in periodontal pockets of the same tooth. In some teeth, unique bacteria in root canals had not passed from periodontal pockets. A basic local alignment search tool (BLAST) sequence search in Genbank indicated that new bacteria species were present in periodontal pockets and root canals. Their characteristics must thus be further analyzed.

  10. 16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances.

    Science.gov (United States)

    Piwat, S; Teanpaisan, R

    2013-01-01

    This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances.

  11. Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Tanaka, Yasushi; Watanabe, Jun; Mogi, Yoshinobu

    2012-08-01

    Soy sauce is a traditional seasoning produced through the fermentation of soybeans and wheat using microbes. In this study, the microbial communities involved in the soy sauce manufacturing process were analyzed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The bacterial DGGE profile indicated that the bacterial microbes in the koji were Weissella cibaria (Weissella confusa, Weissella kimchii, Weissella salipiscis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus iners, or Streptococcus thermophilus), Staphylococcus gallinarum (or Staphylococcus xylosus), and Staphylococcus kloosii. In addition to these bacteria, Tetragenococcus halophilus was also detected in the mash during lactic acid fermentation. The fungal DGGE profile indicated that the fungal microbes in the koji were not only Aspergillus oryzae but also several yeasts. In the mash, Zygosaccharomyces rouxii appeared in the early fermentation stage, Candida etchellsii (or Candida nodaensis) and Candida versatilis were detected at the middle fermentation stage, and Candida etchellsii was detected at the mature fermentation stage. These results suggest that the microbial communities present during the soy sauce manufacturing process change drastically throughout its production. This is the first report to reveal the microbial communities involved in the soy sauce manufacturing process using a culture-independent method. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  12. Analysis of microbial diversity on deli slicers using polymerase chain reaction and denaturing gradient gel electrophoresis technologies.

    Science.gov (United States)

    Koo, O K; Mertz, A W; Akins, E L; Sirsat, S A; Neal, J A; Morawicki, R; Crandall, P G; Ricke, S C

    2013-02-01

    Cross-contamination of pathogenic and spoilage bacteria from food-contact surfaces to food products is a serious public health issue. Bacteria may survive and attach to food-contact surfaces by residual food components and/or background bacteria which may subsequently transfer to other food products. Deli slicers, generally used for slicing ready-to-eat products, can serve as potential sources for considerable bacterial transfer. The objective of this study was to assess the extent and distribution of microbial diversity of deli slicers by identification of pathogenic and background bacteria. Slicer-swab samples were collected from restaurants in Arkansas and Texas in the United States. Ten surface areas for each slicer were swabbed using sterile sponges. Denaturing gradient gel electrophoresis (DGGE) was applied to investigate the fingerprint of samples, and each band was further identified by sequence analysis. Pseudomonads were identified as the dominant bacteria followed by Enterobacteriaceae family, and lactic acid bacteria such as Lactococcus lactis and Streptococcus thermophilus were also found. Bacterial distribution was similar for all surface areas, while the blade guard exhibited the greatest diversity. This study provides a profile of the microbial ecology of slicers using DGGE to develop more specific sanitation practices and to reduce cross-contamination during slicing. © 2012 The Society for Applied Microbiology.

  13. Denaturing gradient gel electrophoresis (DGGE as a powerful novel alternative for differentiation of epizootic ISA virus variants.

    Directory of Open Access Journals (Sweden)

    Marisela Carmona

    Full Text Available Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE, which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens.

  14. Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil.

    Science.gov (United States)

    Jousset, Alexandre; Lara, Enrique; Nikolausz, Marcell; Harms, Hauke; Chatzinotas, Antonis

    2010-02-01

    Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats. Copyright 2009 Elsevier B.V. All rights reserved.

  15. A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC and the Identification of 9 New Mutations Previously Unidentified by DGGE

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff J

    2003-12-01

    Full Text Available Abstract Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE with denaturing high performance liquid chromatography (DHPLC in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE. From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

  16. Behavior of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Ercolini, D; Moschetti, G; Blaiotta, G; Coppola, S

    2001-03-01

    Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

  17. Monomorphism in humans and sequence differences among higher primates for a sequence tagged site (STS) in homeo box cluster 2 as assayed by denaturing gradient electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Ruano, G.; Ruddle, F.H.; Kidd, K.K. (Yale Univ., New Haven, CT (United States)); Gray, M.R. (Tufts Univ., Boston, MA (United States)); Miki, Tetsuro (Osaka Univ. (Japan)); Ferguson-Smith, A.C. (Inst. of Animal Physiology and Genetics Research, Cambridge (United Kingdom))

    1990-03-11

    The human homeo box cluster 2 (HOX2) contains genes coding for DNA binding proteins involved in developmental control and is highly conserved between mouse and man. The authors have applied in concert the Polymerase Chain Reaction (PCR) and Denaturing Gradient Electrophoresis (DGE) to amplify defined primate HOX2 segments and to detect sequence differences among them. They have sequenced a PstI fragment 4 kb upstream from HOX 2.2 and synthesized primers delimiting both halves of 630 bp segment within it PCR on various unrelated humans and SC-PCR on chimpanzee, gorilla, orangutan and gibbon yielded products of the same length for each primer pair.

  18. Chromatin structure influence of DNA damage measurements by four assays: pulsed- and constant-field gel electrophoresis, DNA precipitation and non-denaturing filter elution

    International Nuclear Information System (INIS)

    Wlodek, D.; Olive, P.L.

    1996-01-01

    The of elution of DNA during non-denaturing filter elution (NFE) often correlates with cell sensitivity to radiation. The elution rate is influenced by two cellular factors: chromatin structure and the number of DNA-strand breaks (DSBs) produced in an intact cell by ionizing radiation. To determine which of the above factors is relevant to cell radiosensitivity, four assays were used to measure induction of DNA damage in three cell lines varying in radiosensitivity (V79, CHO, and L5178Y-R). Each of the assays, neutral filter elution (NFE), DNA precipitation, constant (CFGE) and pulsed field gel electrophoresis (PFGE) have different physical basis for DNA damage measurement and might be differently affected by chromatin structure. Three of the methods used to measure DNA double-strand breaks gave different results: NFE was dependent on cell type and location of DNA relative to the replication fork, gel electrophoresis was independent of cell type but was affected by proximity to the replication fork, and the precipitation assay was independent of both cell type and replication status. Pulsed field gel electrophoresis produced the same results and constant field gel electrophoresis for 3 cell lines examined. Only NFE showed differences in sensitivity which correlated with cell survival following irradiation. The results suggest that three is the same initial amount of DSBs in cells from all three lines and that the sensitivity to radiation is determined by some additional factors, probably chromatin structure. (author). 18 refs, 5 figs

  19. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches

    Science.gov (United States)

    Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

    2017-01-01

    Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples. PMID:28955365

  20. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches

    Directory of Open Access Journals (Sweden)

    Xiasheng Zheng

    2017-09-01

    Full Text Available Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis, to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control.Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

  1. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches.

    Science.gov (United States)

    Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

    2017-01-01

    Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight : We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

  2. In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Bello, F D; Walter, J; Hertel, C; Hammes, W P

    2001-07-01

    Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.

  3. Culture-based and denaturing gradient gel electrophoresis analysis of the bacterial community from Chungkookjang, a traditional Korean fermented soybean food.

    Science.gov (United States)

    Hong, Sung Wook; Choi, Jae Young; Chung, Kun Sub

    2012-10-01

    The bacterial community of Chungkookjang and raw rice-straw collected from various areas in South Korea was investigated using both culture-dependent and culture-independent methods. Pure cultures were isolated from Chungkookjang and raw rice-straw on tryptic soy agar plates with 72 to 121 colonies and identified by 16S rDNA gene sequence analysis, respectively. The traditional culture-based method and denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rDNA confirmed that Pantoea agglomerans and B. subtilis were identified as predominant in the raw rice-straw and Chungkookjang, respectively, from Iljuk district of Gyeonggi province, P. ananatis and B. licheniformis were identified as predominant in the raw rice-straw and Chungkookjang from Wonju district of Gangwon province, and Microbacterium sp. and B. licheniformis were identified as predominant in the raw rice-straw and Chungkookjang from Sunchang district of Jeolla province. Other strains, such as Bacillus, Enterococcus, Pseudomonas, Rhodococcus, and uncultured bacteria were also present in raw rice-straw and Chungkookjang. A comprehensive analysis of these microorganisms would provide a more detailed understanding of the biologically active components of Chungkookjang and help improve its quality. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis can be successfully applied to a fermented food to detect unculturable or more species than the culture-dependent method. This technique is an effective and convenient culture-independent method for studying the bacterial community in Chungkookjang. In this study, the bacterial community of Chungkookjang collected from various areas in South Korea was investigated using both culture-dependent and culture-independent methods. © 2012 Institute of Food Technologists®

  4. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

    2009-11-15

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  5. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Science.gov (United States)

    Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

    2009-11-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  6. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    International Nuclear Information System (INIS)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard; Solari, Pier Lorenzo; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis

    2009-01-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  7. Detection in a Japanese population of a length polymorphism in the 5' flanking region of the human β-globin gene with denaturing gradient gel electrophoresis

    International Nuclear Information System (INIS)

    Takahashi, Noria; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko

    1992-10-01

    An analysis of the ATTTT repeat polymorphism located approximately 1,400 base pairs upstream from the β-globin structural gene was carried out by denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes. Genomic or cloned DNAs were digested with restriction enzymes and hybridized with 32 P-labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. A difference in the number of repeat units was recognized by differences in duplex mobility on the DGGE gel. In this study of 81 unrelated Japanese from Hiroshima, a sequence heteromorphism was observed at this site. Alleles with 5 and 6 repeats of the ATTTT unit, which had already been reported, were found in polymorphic proportions. In addition, two unreported alleles, one having 7 repeats and the other having an A-to-G nucleotide substitution in the 5th repeat, were detected. Family study data showed that the segregation of these four types of variants is consistent with an autosomal codominant mode of inheritance. This study also demonstrated that DGGE of RNA:DNA duplexes is a sensitive tool for detecting variations in DNA. (author)

  8. Abundance and Diversity of Hydrogenotrophic Microorganisms in the Infant Gut before the Weaning Period Assessed by Denaturing Gradient Gel Electrophoresis and Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Valeria Sagheddu

    2017-06-01

    Full Text Available Delivery mode (natural vs. cesarean and feeding type (breast vs. formula feeding are relevant factors for neonatal gut colonization. Biomolecular methods have shown that the ecological structure of infant microbiota is more complex than previously proposed, suggesting a relevant presence of unculturable bacteria. It has also been postulated that among unculturable bacteria, hydrogenotrophic populations might play a key role in infant health. Sulfate-reducing bacteria (SRB, acetogens, and methanogenic archaea use hydrogenotrophic pathways within the human colon. However, to date, few studies have reported detection of hydrogenotrophic microorganisms in newborns, possibly because of limitations on available group-specific, culture-independent quantification procedures. In the present work, we analyzed 16 fecal samples of healthy babies aged 1–6 months by means of quantitative PCR (qPCR targeting the 16S rRNA or metabolic functional genes and by denaturing gradient gel electrophoresis (DGGE. qPCR data showed quantifiable levels of methanogens, SRB, and acetogens in all samples, indicating that the relative abundances of these microbial groups were not affected by delivery mode (natural vs. caesarian. DGGE revealed a high prevalence of the Blautia genus within the acetogenic bacteria despite strong interindividual variability. Our preliminary results suggest that hydrogenotrophic microorganisms, which have been a neglected group to date, should be included in future ecological and metabolic studies evaluating the infant intestinal microbiota.

  9. Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Haruta, Shin; Ueno, Shintaro; Egawa, Isao; Hashiguchi, Kazunori; Fujii, Akira; Nagano, Masanobu; Ishii, Masaharu; Igarashi, Yasuo

    2006-05-25

    Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.

  10. Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR▿

    Science.gov (United States)

    Vitali, Beatrice; Pugliese, Ciro; Biagi, Elena; Candela, Marco; Turroni, Silvia; Bellen, Gert; Donders, Gilbert G. G.; Brigidi, Patrizia

    2007-01-01

    The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA. PMID:17644631

  11. Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR.

    Science.gov (United States)

    Vitali, Beatrice; Pugliese, Ciro; Biagi, Elena; Candela, Marco; Turroni, Silvia; Bellen, Gert; Donders, Gilbert G G; Brigidi, Patrizia

    2007-09-01

    The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

  12. Bacterial Diversity in the Digestive Tracts of Four Indian Air-Breathing Fish Species Investigated by PCR Based Denaturing Gradient Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Suxu He

    Full Text Available ABSTRACT An investigation was conducted to identify the allochthonous microbiota (entire intestine and the autochthonous microbiota in proximal intestine (PI and distal intestine (DI of four species of Indian air-breathing fish (climbing perch; Anabas testudineus, murrel; Channa punctatus, walking catfish; Clarias batrachus and stinging catfish; Heteropneustes fossilis by PCR based denaturing gradient gel electrophoresis (DGGE. High similarities of the allochthonous microbiota were observed between climbing perch and murrel, walking catfish and stinging catfish, indicating similar food behavior. The autochthonous microbiota of PI and DI from climbing perch and murrel revealed more similarity, than the result obtained from walking catfish and stinging catfish. The autochthonous microbiota of climbing perch and murrel were similar with regard to the allochthonous microbiota, but no such similarity was observed in case of walking catfish and stinging catfish. The fish genotype and intestinal bacteria are well matched and show co-evolutionary relationship. Three fish species has its unique bacteria; autochthonous Enterobacter cloacae, Edwardsiella tarda and Sphingobium sp. in DI of climbing perch, Pseudomonas sp.; allochthonous and autochthonous in PI of walking catfish and uncultured bacterium (EU697160.1, uncultured bacterium (JF018065.1 and uncultured bacterium (EU697160.1 for stinging catfish. In murrel, no unique bacteria were detected.

  13. Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing.

    Science.gov (United States)

    Saraithong, Prakaimuk; Li, Yihong; Saenphet, Kanokporn; Chen, Zhou; Chantawannakul, Panuwan

    2015-10-01

    This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol-chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  14. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    Science.gov (United States)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  15. Denaturing Gradient Gel Electrophoresis-Polymerase Chain Reaction Comparison of Chitosan Effects on Anaerobic Cultures of Broiler Cecal Bacteria and Salmonella Typhimurium.

    Science.gov (United States)

    Hume, Michael; Sohail, Muhammad Umar

    2018-04-01

    Enteropathogen colonization and product contamination are major poultry industry problems. The emergence of antibiotic resistance, and associated risks to human health, is limiting the use of antibiotics as first-line defense against enteropathogens in poultry. The chitin derivative, chitosan, has drawn substantial attention for its bactericidal properties. Different molecular weight (MW) chitosans can have varied effects against different bacteria in monoculture. In the current study, cecal contents from each of three market-age broilers and Salmonella Typhimurium, as indicator enteropathogen, were exposed to in vitro anaerobic culture to three chitosan preparations (0.08%, wt/vol), low (LMW), medium (MMW), and coarse (CMW). Effects of chitosan and the carrier solvent acetic acid, on cecal bacteria and Salmonella, were examined by denaturing gradient gel electrophoresis (DGGE) and Salmonella enumeration. Bacterial profiles for the three cecal contents were shown by DGGE to be very different. Each of the three cecal contents grown in the presence of 0.08% acetic acid was very different from the same contents grown without the chitosan solvent. Culturing cecal contents in the presence of chitosan altered the bacterial DGGE profiles from the control and acetic acid-only cultures. The DGGE chitosan-treated profiles for all three cecal sources were identical to each other regardless of the MW chitosan in the culture medium. Compared with Salmonella in monoculture, Salmonella decreased (p < 0.05) by about 1.5 log CFU/mL when grown in mixed culture with cecal contents. Salmonella monocultures in the presence of 0.08% of the chitosan solvent acetic acid decreased (p < 0.05) counts by almost 3.5 log CFU/mL. Combining acetic acid and cecal contents reduced (p < 0.05) Salmonella by 7 log CFU/mL. Adding the chitosan preparations to the mixtures reduced (p < 0.05) Salmonella by 8 log CFU/mL.

  16. Application of PCR-denaturing-gradient gel electrophoresis (DGGE) method to examine microbial community structure in asparagus fields with growth inhibition due to continuous cropping.

    Science.gov (United States)

    Urashima, Yasufumi; Sonoda, Takahiro; Fujita, Yuko; Uragami, Atsuko

    2012-01-01

    Growth inhibition due to continuous cropping of asparagus is a major problem; the yield of asparagus in replanted fields is low compared to that in new fields, and missing plants occur among young seedlings. Although soil-borne disease and allelochemicals are considered to be involved in this effect, this is still controversial. We aimed to develop a technique for the biological field diagnosis of growth inhibition due to continuous cropping. Therefore, in this study, fungal community structure and Fusarium community structure in continuously cropped fields of asparagus were analyzed by polymerase chain reaction/denaturing-gradient gel electrophoresis (PCR-DGGE). Soil samples were collected from the Aizu region of Fukushima Prefecture, Japan. Soil samples were taken from both continuously cropped fields of asparagus with growth inhibition and healthy neighboring fields of asparagus. The soil samples were collected from the fields of 5 sets in 2008 and 4 sets in 2009. We were able to distinguish between pathogenic and non-pathogenic Fusarium by using Alfie1 and Alfie2GC as the second PCR primers and PCR-DGGE. Fungal community structure was not greatly involved in the growth inhibition of asparagus due to continuous cropping. By contrast, the band ratios of Fusarium oxysporum f. sp. asparagi in growth-inhibited fields were higher than those in neighboring healthy fields. In addition, there was a positive correlation between the band ratios of Fusarium oxysporum f. sp. asparagi and the ratios of missing asparagus plants. We showed the potential of biological field diagnosis of growth inhibition due to continuous cropping of asparagus using PCR-DGGE.

  17. Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection.

    Science.gov (United States)

    Hume, Michael E; Barbosa, Nei A; Dowd, Scot E; Sakomura, Nilva K; Nalian, Armen G; Martynova-Van Kley, Alexandra; Oviedo-Rondón, Edgar O

    2011-11-01

    A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in

  18. Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity.

    Science.gov (United States)

    Saro, Cristina; Molina-Alcaide, Eduarda; Abecia, Leticia; Ranilla, María José; Carro, María Dolores

    2018-04-01

    The objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.

  19. Capillaries modified by noncovalent anionic polymer adsorption for capillary zone electrophoresis, micellar electrokinetic capillary chromatography and capillary electrophoresis mass spectrometry

    DEFF Research Database (Denmark)

    Bendahl, L; Hansen, S H; Gammelgaard, Bente

    2001-01-01

    A simple coating procedure for generation of a high and pH-independent electroosmotic flow in capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) is described. The bilayer coating was formed by noncovalent adsorption of the ionic polymers Polybrene...... capillaries was (4.9+/-0.1) x 10(-4) cm2V(-1)s(-1) in a pH-range of 2-10 (ionic strength = 30 mM). When alkaline compounds were used as test substances intracapillary and intercapillary migration time variations (n = 6) were less than 1% relative standard deviation (RSD) and 2% RSD, respectively in the entire...... pH range. The coating was fairly stable in the presence of sodium dodecyl sulfate, and this made it possible to perform fast MEKC separations at low pH. When neutral compounds were used as test substances, the intracapillary migration time variations (n = 6) were less than 2% RSD in a pH range of 2...

  20. Evaluation of different PCR primers for denaturing gradient gel electrophoresis (DGGE) analysis of fungal community structure in traditional fermentation starters used for Hong Qu glutinous rice wine.

    Science.gov (United States)

    Lv, Xu-Cong; Jiang, Ya-Jun; Liu, Jie; Guo, Wei-Ling; Liu, Zhi-Bin; Zhang, Wen; Rao, Ping-Fan; Ni, Li

    2017-08-16

    Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial community structure. However, when DGGE is applied to evaluate the fungal community of traditional fermentation starters, the choice of hypervariable ribosomal RNA gene regions is still controversial. In the current study, several previously published fungal PCR primer sets were compared and evaluated using PCR-DGGE, with the purpose of screening a suitable primer set to study the fungal community of traditional fermentation starters for Hong Qu glutinous rice wine. Firstly, different primer sets were used to amplify different hypervariable regions from pure fungal cultures. Except NS1/FR1+ and ITS1fGC/ITS4, other primer sets (NL1+/LS2R, NL3A/NL4GC, FF390/FR1+, NS1/GCFung, NS3+/YM951r and ITS1fGC/ITS2r) amplified the target DNA sequences successfully. Secondly, the selected primer sets were further evaluated based on their resolution to distinguish different fungal cultures through DGGE fingerprints. Three primer sets (NL1+/LS2R, NS1/GCFung and ITS1fGC/ITS2r) were finally selected for investigating the fungal community structure of different traditional fermentation starters for Hong Qu glutinous rice wine. The internal transcribed spacer (ITS) region amplified by ITS1fGC/ITS2r, which is more hypervariable than the 18S rRNA gene and 26S rRNA gene, provides an excellent tool to separate amplification products of different fungal species. Results indicated that PCR-DGGE profile using ITS1fGC/ITS2r showed more abundant fungal species than that using NL1+/LS2R and NS1/GCFung. Therefore, ITS1fGC/ITS2r is the most suitable primer set for PCR-DGGE analysis of fungal community structure in traditional fermentation starters for Hong Qu glutinous rice wine. DGGE profiles based on ITS1fGC/ITS2r revealed the presence of twenty-four fungal species in traditional fermentation starter. A significant difference of fungal community can be observed directly from DGGE fingerprints and

  1. A novel polymerase chain reaction (PCR) - denaturing gradient gel electrophoresis (DGGE) for the identification of Micrococcaceae strains involved in meat fermentations. Its application to naturally fermented Italian sausages.

    Science.gov (United States)

    Cocolin, L; Manzano, M; Aggio, D; Cantoni, C; Comi, G

    2001-05-01

    A new molecular method consisting of polymerase chain reaction (PCR) amplification and denaturing gradient gel electrophoresis (DGGE) of a small fragment from the 16S rRNA gene identified the Micrococcaceae strains isolated from natural fermented Italian sausages. Lactic acid bacteria, total aerobic mesophilic flora, Enterobacteriaceae and faecal enterococci were also monitored. Micrococcaceaea control strains from international collections were used to optimise the method and 90 strains, isolated from fermented sausages, were identified by biochemical tests and PCR-DGGE. No differences were observed between the methods used. The results reported in this paper prove that Staphylococcus xylosus is the main bacterium involved in fermented sausage production, representing, from the tenth day of ripening, the only Micrococcaceaea species isolated.

  2. Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet.

    Science.gov (United States)

    Musyimi, Harrison K; Guy, Jason; Narcisse, Damien A; Soper, Steven A; Murray, Kermit K

    2005-12-01

    We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.

  3. Effect of surfactant species and electrophoretic medium composition on the electrophoretic behavior of neutral and water-insoluble linear synthetic polymers in nonaqueous capillary zone electrophoresis.

    Science.gov (United States)

    Fukai, Nao; Kitagawa, Shinya; Ohtani, Hajime

    2017-07-01

    We have recently demonstrated the separation of neutral and water-insoluble linear synthetic polymers in nonaqueous capillary zone electrophoresis (NACZE) using a cationic surfactant of cetyltrimethylammonium chloride (CTAC). In this study, eight ionic surfactants were investigated for the separation of four synthetic polymers (polystyrene, polymethylmethacrylates, polybutadiene, and polycarbonate); only three surfactants (CTAC, dimethyldioctadecylammonium bromide, and sodium dodecylsulfate) caused their separation. The order of the interaction between the polymers and the surfactants depended on both the surfactant species and the composition of the electrophoretic medium. Their investigation revealed that the separation is majorly affected by the hydrophobic interactions between the polymers and the ionic surfactants. In addition, the electrophoretic behavior of polycarbonate suggested that electrostatic interaction also affects the selectivity of the polymers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 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

  5. Denatured fuel cycles

    International Nuclear Information System (INIS)

    Till, C.E.

    1979-01-01

    This paper traces the history of the denatured fuel concept and discusses the characteristics of fuel cycles based on the concept. The proliferation resistance of denatured fuel cycles, the reactor types they involve, and the limitations they place on energy generation potential are discussed. The paper concludes with some remarks on the outlook for such cycles

  6. Two-photon excitation in chip electrophoresis enabling label-free fluorescence detection in non-UV transparent full-body polymer chips.

    Science.gov (United States)

    Geissler, David; Belder, Detlev

    2015-12-01

    One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep-ultraviolet (UV) excitation (electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Hand-held optical sensor using denatured antibody coated electro-active polymer for ultra-trace detection of copper in blood serum and environmental samples.

    Science.gov (United States)

    Chandra, Sutapa; Dhawangale, Arvind; Mukherji, Soumyo

    2018-07-01

    An optimum copper concentration in environment is highly desired for all forms of life. We have developed an ultrasensitive copper sensor which functions from femto to micro molar concentration accurately (R 2 = 0.98). The sensor is based on denatured antibody immunoglobulin G (IgG), immobilized on polyaniline (PAni) which in turn is the coating on the core of an optical fiber. The sensing relies on changes in evanescent wave absorbance in the presence of the analyte. The sensor showed excellent selectivity towards Cu (II) ions over all other metal ions. The sensor was tested with lake and marine water samples to determine unknown concentrations of copper ions and the recovery results were within 90-115%, indicating reasonable accuracy. We further integrated the fiber-optic sensor with a miniaturized hand-held instrumentation platform to develop an accurate and field deployable device which can broadly be applicable to determine Cu (II) concentration in a wide range of systems - natural water bodies, soil as well as blood serum. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Mutation screening of the TP53 gene by temporal temperature gradient gel electrophoresis.

    Science.gov (United States)

    Sørlie, Therese; Johnsen, Hilde; Vu, Phuong; Lind, Guro Elisabeth; Lothe, Ragnhild; Børresen-Dale, Anne-Lise

    2005-01-01

    A protocol for detection of mutations in the TP53 gene using temporal temperature gradient gel electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) and eliminates some of the problems. The result is a rapid and sensitive screening technique that is robust and easily set up in smaller laboratory environments.

  9. Mutation screening of the TP53 gene by temporal temperature gel electrophoresis (TTGE).

    Science.gov (United States)

    Sørlie, Therese; Johnsen, Hilde; Vu, Phuong; Lind, Guro Elisabeth; Lothe, Ragnhild; Børresen-Dale, Anne-Lise

    2014-01-01

    A protocol for detection of mutations in the TP53 gene using temporal temperature gradient electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques, denaturing gradient gel electrophoresis and constant denaturant gel electrophoresis, and eliminates some of the problems. The result is a rapid and sensitive screening technique which is robust and easily set up in smaller laboratory environments.

  10. Separation of plant hormones from biofertilizer by capillary electrophoresis using a capillary coated dynamically with polycationic polymers.

    Science.gov (United States)

    Jiang, Ting-Fu; Lv, Zhi-Hua; Wang, Yuan-Hong; Yue, Mei-E

    2006-06-01

    A new, simple and rapid capillary electrophoresis (CE) method, using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier, was developed for the identification and quantitative determination of four plant hormones, including gibberellin A3 (GA3), indole-3-acetic acid (IAA), alpha-naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CA). The optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.005% (w/v) of HDB. The applied voltage was -25 kV and the capillary temperature was kept constant at 25 degrees C. Salicylic acid was used as internal standard for quantification. The calibration dependencies exhibited good linearity within the ratios of the concentrations of standard samples and internal standard and the ratios of the peak areas of samples and internal standard. The correlation coefficients were from 0.9952 to 0.9997. The relative standard deviations of migration times and peak areas were biofertilizer were successfully determined within 7 min, with satisfactory repeatability and recovery.

  11. Electrophoresis technology

    Science.gov (United States)

    Snyder, R. S.

    1985-01-01

    A new high resolution apparatus designed for space was built as a laboratory prototype. Using a moving wall with a low zeta potential coating, the major sources of flow distortion for an electrophoretic sample stream are removed. Highly resolved fractions, however, will only be produced in space because of the sensitivity of this chamber to buoyancy-induced convection in the laboratory. The second and third flights of the McDonnell Douglas Astronautics Corporation continuous flow electrophoresis system carried samples developed at MSFC intended to evaluate the broad capabilities of free flow electrophoresis in a reduced gravity environment. Biological model materials, hemoglobin and polystyrene latex microspheres, were selected because of their past use as electrophoresis standards and as visible markers for fluid flow due to electroosmosis, spacecraft acceleration or other factors. The dependence of the separation resolution on the properties of the sample and its suspension solution was assessed.

  12. Analysis of urinary neurotransmitters by capillary electrophoresis: Sensitivity enhancement using field-amplified sample injection and molecular imprinted polymer solid phase extraction

    International Nuclear Information System (INIS)

    Claude, Berengere; Nehme, Reine; Morin, Philippe

    2011-01-01

    Highlights: → Field-amplified sample injection (FASI) improves the sensitivity of capillary electrophoresis through the online pre-concentration samples. → The cationic analytes are stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. → The limits of quantification are 500 times lower than those obtained with hydrodynamic injection. → The presence of salts in the matrix greatly reduces the sensitivity of the FASI/CE-UV method. - Abstract: Capillary electrophoresis (CE) has been investigated for the analysis of some neurotransmitters, dopamine (DA), 3-methoxytyramine (3-MT) and serotonin (5-hydroxytryptamine, 5-HT) at nanomolar concentrations in urine. Field-amplified sample injection (FASI) has been used to improve the sensitivity through the online pre-concentration samples. The cationic analytes were stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. Several FASI parameters have been optimized (ionic strength of the running buffer, concentration of the sample protonation agent, composition of the sample solvent and nature of the pre-injection plug). Best results were obtained using H 3 PO 4 -LiOH (pH 4, ionic strength of 80 mmol L -1 ) as running buffer, 100 μmol L -1 of H 3 PO 4 in methanol-water 90/10 (v/v) as sample solvent and 100 μmol L -1 of H 3 PO 4 in water for the pre-injection plug. In these conditions, the linearity was verified in the 50-300 nmol L -1 concentration range for DA, 3-MT and 5-HT with a determination coefficient (r 2 ) higher than 0.99. The limits of quantification (10 nmol L -1 for DA and 3-MT, 5.9 nmol L -1 for 5-HT) were 500 times lower than those obtained with hydrodynamic injection. However, if this method is applied to the analysis of neurotransmitters in urine, the presence of salts in the matrix greatly reduces the sensitivity

  13. Analysis of urinary neurotransmitters by capillary electrophoresis: Sensitivity enhancement using field-amplified sample injection and molecular imprinted polymer solid phase extraction

    Energy Technology Data Exchange (ETDEWEB)

    Claude, Berengere, E-mail: berengere.claude@univ-orleans.fr [Institut de Chimie Organique et Analytique, CNRS FR 2708 UMR 6005, Universite d' Orleans, 45067 Orleans (France); Nehme, Reine; Morin, Philippe [Institut de Chimie Organique et Analytique, CNRS FR 2708 UMR 6005, Universite d' Orleans, 45067 Orleans (France)

    2011-08-12

    Highlights: {yields} Field-amplified sample injection (FASI) improves the sensitivity of capillary electrophoresis through the online pre-concentration samples. {yields} The cationic analytes are stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. {yields} The limits of quantification are 500 times lower than those obtained with hydrodynamic injection. {yields} The presence of salts in the matrix greatly reduces the sensitivity of the FASI/CE-UV method. - Abstract: Capillary electrophoresis (CE) has been investigated for the analysis of some neurotransmitters, dopamine (DA), 3-methoxytyramine (3-MT) and serotonin (5-hydroxytryptamine, 5-HT) at nanomolar concentrations in urine. Field-amplified sample injection (FASI) has been used to improve the sensitivity through the online pre-concentration samples. The cationic analytes were stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. Several FASI parameters have been optimized (ionic strength of the running buffer, concentration of the sample protonation agent, composition of the sample solvent and nature of the pre-injection plug). Best results were obtained using H{sub 3}PO{sub 4}-LiOH (pH 4, ionic strength of 80 mmol L{sup -1}) as running buffer, 100 {mu}mol L{sup -1} of H{sub 3}PO{sub 4} in methanol-water 90/10 (v/v) as sample solvent and 100 {mu}mol L{sup -1} of H{sub 3}PO{sub 4} in water for the pre-injection plug. In these conditions, the linearity was verified in the 50-300 nmol L{sup -1} concentration range for DA, 3-MT and 5-HT with a determination coefficient (r{sup 2}) higher than 0.99. The limits of quantification (10 nmol L{sup -1} for DA and 3-MT, 5.9 nmol L{sup -1} for 5-HT) were 500 times lower than those obtained with hydrodynamic injection. However, if this method is applied to the analysis of

  14. 27 CFR 19.456 - Adding denaturants.

    Science.gov (United States)

    2010-04-01

    ... proprietor shall submit a flow diagram of the intended process or method of adding denaturants. (Sec. 201... OF THE TREASURY LIQUORS DISTILLED SPIRITS PLANTS Denaturing Operations and Manufacture of Articles...

  15. 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)

  16. Single DNA denaturation and bubble dynamics

    DEFF Research Database (Denmark)

    Metzler, Ralf; Ambjörnsson, Tobias; Hanke, Andreas

    2009-01-01

    While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration......, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments...... for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation...

  17. Single DNA denaturation and bubble dynamics

    International Nuclear Information System (INIS)

    Metzler, Ralf; Ambjoernsson, Tobias; Hanke, Andreas; Fogedby, Hans C

    2009-01-01

    While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation bubbles and selectively single-stranded DNA binding proteins.

  18. Electrophoresis in strong electric fields.

    Science.gov (United States)

    Barany, Sandor

    2009-01-01

    function of the electric field strength, particle size, electrolyte concentration and the adsorbed polymer amount. It has been shown that the electrophoretic velocity of the particles/cells increases with field strength linearly up to about 100 and 200 V/cm (for cells) without and with adsorbed polymers both in pure water and in electrolyte solutions. In line with the theoretical predictions, in stronger fields substantial non-linear effects were recorded (V(ef)~E(3)). The ef velocity of unipolar ion-type conducting (ion-exchanger particles and fibres), electron-type conducting (magnesium and Mg/Al alloy) and semiconductor particles (graphite, activated carbon, pyrite, molybdenite) increases significantly with the electric field (V(ef)~E(2)) and the particle's size but is almost independent of the ionic strength. These trends are inconsistent with Smoluchowski's equation for dielectric particles, but are consistent with the Dukhin-Mishchuk theory of superfast electrophoresis.

  19. Electrophoresis forum '80

    International Nuclear Information System (INIS)

    Radola, B.J.

    1980-01-01

    In this volume the contributions of the electrophoresis meeting are presented in a short term form. The main topics are gel-electrophoresis, ultra thin film isoelectric focusing, one- and two-dimensional electrophoresis, electrophoretical separation techniques, electric focusing (for phorensic studies), substrate free and substrate electrophoresis. In the poster session of this meeting subjects such as (ultra) thin film isoelectric focusing, identification of radioactive proteins, labelling of cell surfaces, autoradiography and 3 H-labelled proteins. Separate abstracts were prepared for 4 papers in this report. (HK) [de

  20. Affinity in electrophoresis.

    Science.gov (United States)

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  1. 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...

  2. Reversibility of partial denaturation of DNA

    International Nuclear Information System (INIS)

    Acuna, M.I.; Mingot, F.; Davila, C.A.

    1976-01-01

    The recovery of hypochromicity in a partially denatured DNA sample when salt concentration is suddenly increased at a intermediate stage of the thermal transition is studied. The results of CsCl gradient analysis, PEG/DEX partition analysis, behaviour in a new thermal transition hydrodynamic properties and transforming ability, support the view that the process is an intramolecular double chain denaturation. The degree of denaturation irreversibility is dependent on single chain molecular weight of DNA (discontinuities denisty) and upon the helicity value at which salt concentration jump is performed. Both dependences are formally interpreted according to Elton's model for base distribution in DNA. Kinetically the process behaves as being an hydrodynamically limited rewinding. (author)

  3. 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 ...

  4. Urine protein electrophoresis test

    Science.gov (United States)

    Urine protein electrophoresis; UPEP; Multiple myeloma - UPEP; Waldenström macroglobulinemia - UPEP; Amyloidosis - UPEP ... special paper and apply an electric current. The proteins move and form visible bands. These reveal the ...

  5. Automated DNA electrophoresis, hybridization and detection

    International Nuclear Information System (INIS)

    Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

    1986-01-01

    A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; 32 P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing

  6. A neutral glyoxal gel electrophoresis method for the detection and semi-quantitation of DNA single-strand breaks.

    Science.gov (United States)

    Pachkowski, Brian; Nakamura, Jun

    2013-01-01

    Single-strand breaks are among the most prevalent lesions found in DNA. Traditional electrophoretic methods (e.g., the Comet assay) used for investigating these lesions rely on alkaline conditions to denature DNA prior to electrophoresis. However, the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Herein, we describe a neutral glyoxal gel electrophoresis assay which is based on alkali-free DNA denaturation and is suitable for qualitative and semi-quantitative analyses of single-strand breaks in DNA isolated from different organisms.

  7. Is pulsed electric field still effective for RNA separation in capillary electrophoresis?

    Science.gov (United States)

    Li, Zhenqing; Dou, Xiaoming; Ni, Yi; Chen, Qinmiao; Cheng, Shuyi; Yamaguchi, Yoshinori

    2012-03-16

    Pulsed field capillary electrophoresis (PFCE) is a predominant technique to cope with difficulties in resolving large DNA strands, yet it is still unclear whether pulsed electric field is effective for the separation of higher mass RNA. In this paper we focused on the role of pulsed electric field in large RNA fragments analysis by comparing RNA separation performance in PFCE with that in constant field CE. Separation performance in terms of migration mobility, plate numbers, resolution, and selectivity has been tested for the analysis of RNA from 0.1 to 10.0 kilo nucleotide (knt) under different electrophoretic conditions. Denaturation, important to obtain uniform and identifiable peaks, was accomplished by heating the sample in 4.0M urea prior to analysis and the presence of 4.0M urea in the electrophoresis buffer. Results demonstrate that unlike DNA in PFCE, the pulsed electric field mainly affects the separation performance of RNA between 0.4 and 2.0 knt. The migration mobility of long RNA fragments is not a strong function of modulation depth and pulsed frequency. Moreover, the logarithm of RNA mobility is almost inversely proportional to the logarithm of molecule size up to 6.0 knt with correlation coefficient higher than 0.99 in all the polymer concentrations measured here. Resonance frequency of RNA in PFCE was also observed. While these initial experiments show no distinct advantages of using PFCE for RNA separation, they do take further step toward characterizing the migration behavior of RNA under pulsed field conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. 9 CFR 325.13 - Denaturing procedures.

    Science.gov (United States)

    2010-01-01

    ... the appropriate agent shall be used to give the material a distinctive color, odor, or taste so that... thoroughly mixing therein denaturing oil, No. 2 fuel oil, brucine dissolved in a mixture of alcohol and pine... distinctive a color, odor, or taste that it cannot be confused with an article of human food. [35 FR 15605...

  9. Effect of heating strategies on whey protein denaturation--Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry.

    Science.gov (United States)

    Akkerman, M; Rauh, V M; Christensen, M; Johansen, L B; Hammershøj, M; Larsen, L B

    2016-01-01

    Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. 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.

  11. Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins.

    Science.gov (United States)

    Sherman, Eilon; Itkin, Anna; Kuttner, Yosef Yehuda; Rhoades, Elizabeth; Amir, Dan; Haas, Elisha; Haran, Gilad

    2008-06-01

    Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.

  12. Analyzing Protein Denaturation using Fast Differential Scanning Calorimetry

    NARCIS (Netherlands)

    Splinter, R.; Van Herwaarden, A.W.; Iervolino, E.; Vanden Poel, G.; Istrate, D.; Sarro, P.M.

    2012-01-01

    This paper investigates the possibility to measure protein denaturation with Fast Differential Scanning Calorimetry (FDSC). Cancer can be diagnosed by measuring protein denaturation in blood plasma using Differential Scanning Calorimetry (DSC). FDSC can reduce diagnosis time from hours to minutes,

  13. Guanidinium-induced denaturation by breaking of salt bridges

    NARCIS (Netherlands)

    Meuzelaar, H.; Panman, M.R.; Woutersen, S.

    2015-01-01

    Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm+) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm+ can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm+-​induced denaturation of

  14. Hysteresis in pressure-driven DNA denaturation.

    Directory of Open Access Journals (Sweden)

    Enrique Hernández-Lemus

    Full Text Available In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue.

  15. A correlated Walks' theory for DNA denaturation

    International Nuclear Information System (INIS)

    Mejdani, R.

    1994-08-01

    We have shown that by using a correlated Walks' theory for the lattice gas model on a one-dimensional lattice, we can study, beside the saturation curves obtained before for the enzyme kinetics, also the DNA denaturation process. In the limit of no interactions between sites the equation for melting curves of DNA reduces to the random model equation. Thus our leads naturally to this classical equation in the limiting case. (author). 22 refs, 3 figs

  16. Denaturing gradient gel electrophoresis as a fingerprinting method for the analysis of soil microbial communities

    Czech Academy of Sciences Publication Activity Database

    Valášková, Vendula; Baldrian, Petr

    2009-01-01

    Roč. 55, č. 10 (2009), s. 413-423 ISSN 1214-1178 R&D Projects: GA ČR GA526/08/0751; GA MŠk LC06066 Institutional research plan: CEZ:AV0Z50200510 Keywords : DGGE * bacteria * fungi Subject RIV: EE - Microbiology, Virology Impact factor: 0.697, year: 2009

  17. A PCR-denaturing gradient gel electrophoresis (DGGE) approach to assess Fusarium diversity in asparagus

    NARCIS (Netherlands)

    Yergeau, E.; Filion, M.; Vujanovic, V.; St-Arnaud, M.

    2005-01-01

    In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these

  18. Denatured plutonium: a study of deterrent action. Final report

    International Nuclear Information System (INIS)

    Hutchins, B.A.

    1975-07-01

    The safeguarding of nuclear reactor fuel includes physical security methods as well as technological process options. The purpose of this study was to provide a preliminary evaluation of a technological option; the introduction of denaturing as a deterrent to illicit plutonium diversion. Denaturing is accomplished by coextracting some highly-radioactive fission products with the plutonium during reprocessing of spent fuel. The radioactive denaturant is always in companion with the plutonium through all subsequent fuel cycle steps - and serves as a deterrent to diversion or illicit usage of this fissile source. In concept the denaturing approach is simple and straightforward. This report provides a preliminary analysis of denaturing which can be achieved within the framework of present reprocessing technology. The impact of denaturing is indicated by comparison to a conventional (i.e., non-denatured) light water reacter cycle approach

  19. Integration of amperometric sensors for microchip capillary electrophoresis application

    International Nuclear Information System (INIS)

    Dicorato, F; Moore, E; Glennon, J

    2011-01-01

    Capillary electrophoresis is a technique for the separation and analysis of chemical compounds. Techniques adopted from the microchip technology knowledge have led to recent developments of electrophoresis system with integration on microchip. Microchip Capillary Electrophoresis (μCE) systems offer a series of advantages as easy integration for Lab-on-a-chip applications, high performance, portability, speed, minimal solvent and sample requirements. A new technological challenge aims at the development of an economic modular microchip capillary electrophoresis systems using separable and independent units concerning the sensor. In this project we worked on the development of an interchangeable amperometric sensor in order to provide a solution to such electrode passivation and facilitating the use of tailored sensors for specific analyte detection besides. Fluidic chips have been machined from cyclic olefin polymer pallets (Zeonor) using a micro-injection molding machine.

  20. Integration of amperometric sensors for microchip capillary electrophoresis application

    Energy Technology Data Exchange (ETDEWEB)

    Dicorato, F; Moore, E [Life Sciences Interface Group, Tyndall National Institute, Lee Maltings, Dyke Parade, Cork (Ireland); Glennon, J, E-mail: eric.moore@tyndall.ie [Chemistry Department, University College Cork, College Road, Cork (Ireland)

    2011-08-17

    Capillary electrophoresis is a technique for the separation and analysis of chemical compounds. Techniques adopted from the microchip technology knowledge have led to recent developments of electrophoresis system with integration on microchip. Microchip Capillary Electrophoresis ({mu}CE) systems offer a series of advantages as easy integration for Lab-on-a-chip applications, high performance, portability, speed, minimal solvent and sample requirements. A new technological challenge aims at the development of an economic modular microchip capillary electrophoresis systems using separable and independent units concerning the sensor. In this project we worked on the development of an interchangeable amperometric sensor in order to provide a solution to such electrode passivation and facilitating the use of tailored sensors for specific analyte detection besides. Fluidic chips have been machined from cyclic olefin polymer pallets (Zeonor) using a micro-injection molding machine.

  1. Synthesis of hydrogel via click chemistry for DNA electrophoresis.

    Science.gov (United States)

    Finetti, Chiara; Sola, Laura; Elliott, Jim; Chiari, Marcella

    2017-09-01

    This work introduces a novel sieving gel for DNA electrophoresis using a classical click chemistry reaction, the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC), to cross-link functional polymer chains. The efficiency of this reaction provides, under mild conditions, hydrogels with near-ideal network connectivity and improved physical properties. Hydrogel formation via click chemistry condensation of functional polymers does not involve the use of toxic monomers and UV initiation. The performance of the new hydrogel in the separation of double stranded DNA fragments was evaluated in the 2200 TapeStation system, an analytical platform, recently introduced by Agilent that combines the advantages of CE in terms of miniaturization and automation with the simplicity of use of slab gel electrophoresis. The click gel enables addition of florescent dyes prior to electrophoresis with considerable improvement of resolution and separation efficiency over conventional cross-linked polyacrylamide gels. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. 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 ...

  3. 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.

  4. Hemichrome formation during hemoglobin Zurich denaturation

    International Nuclear Information System (INIS)

    Zago, M.A.; Costa, F.F.; Botura, C.; Baffa, O.

    1988-01-01

    Electron paramagnetic resonance (EPR)spectrum of hemoglobin Zurich, after oxidation, storage and heating, showed several absorption derives in the high field region (g ≅ 2) which are indicative of hemichrome formation. Characteristic visible spectra of hemichromes were observed for oxidized Hb Zurich and for its spontaneous precipitate. The proportional increase of EPR signals at g ≅ 2 and decrease at g = 6.37, the constant ratio of absorbance at 540 nm to 280 nm during heating, and the similarity of this ratio for spontaneously precipitated HbA and for Hb Zurich indicate that heme is not lost during the first steps of Hb Zurich denaturation. (author) [pt

  5. Partially folded intermediates during trypsinogen denaturation

    Directory of Open Access Journals (Sweden)

    Martins N.F.

    1999-01-01

    Full Text Available The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

  6. Virus Infection Triggers MAVS Polymers of Distinct Molecular Weight

    Directory of Open Access Journals (Sweden)

    Natalia Zamorano Cuervo

    2018-01-01

    Full Text Available The mitochondrial antiviral signaling (MAVS adaptor protein is a central signaling hub required for cells to mount an antiviral response following virus sensing by retinoic acid-inducible gene I (RIG-I-like receptors. MAVS localizes in the membrane of mitochondria and peroxisomes and in mitochondrial-associated endoplasmic reticulum membranes. Structural and functional studies have revealed that MAVS activity relies on the formation of functional high molecular weight prion-like aggregates. The formation of protein aggregates typically relies on a dynamic transition between oligomerization and aggregation states. The existence of intermediate state(s of MAVS polymers, other than aggregates, has not yet been documented. Here, we used a combination of non-reducing SDS-PAGE and semi-denaturing detergent agarose gel electrophoresis (SDD-AGE to resolve whole cell extract preparations to distinguish MAVS polymerization states. While SDD-AGE analysis of whole cell extracts revealed the formation of previously described high molecular weight prion-like aggregates upon constitutively active RIG-I ectopic expression and virus infection, non-reducing SDS-PAGE allowed us to demonstrate the induction of lower molecular weight oligomers. Cleavage of MAVS using the NS3/4A protease revealed that anchoring to intracellular membranes is required for the appropriate polymerization into active high molecular weight aggregates. Altogether, our data suggest that RIG-I-dependent MAVS activation involves the coexistence of MAVS polymers with distinct molecular weights.

  7. Study of polymer molecules and conformations with a nanopore

    Science.gov (United States)

    Golovchenko, Jene A.; Li, Jiali; Stein, Derek; Gershow, Marc H.

    2010-12-07

    The invention features methods for evaluating the conformation of a polymer, for example, for determining the conformational distribution of a plurality of polymers and to detect binding or denaturation events. The methods employ a nanopore which the polymer, e.g., a nucleic acid, traverses. As the polymer traverses the nanopore, measurements of transport properties of the nanopore yield data on the conformation of the polymer.

  8. Simulated pressure denaturation thermodynamics of ubiquitin.

    Science.gov (United States)

    Ploetz, Elizabeth A; Smith, Paul E

    2017-12-01

    Simulations of protein thermodynamics are generally difficult to perform and provide limited information. It is desirable to increase the degree of detail provided by simulation and thereby the potential insight into the thermodynamic properties of proteins. In this study, we outline how to analyze simulation trajectories to decompose conformation-specific, parameter free, thermodynamically defined protein volumes into residue-based contributions. The total volumes are obtained using established methods from Fluctuation Solution Theory, while the volume decomposition is new and is performed using a simple proximity method. Native and fully extended ubiquitin are used as the test conformations. Changes in the protein volumes are then followed as a function of pressure, allowing for conformation-specific protein compressibility values to also be obtained. Residue volume and compressibility values indicate significant contributions to protein denaturation thermodynamics from nonpolar and coil residues, together with a general negative compressibility exhibited by acidic residues. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Reversible thermal denaturation of immobilized rhodanese

    International Nuclear Information System (INIS)

    Horowitz, P.; Bowman, S.

    1987-01-01

    For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [ 35 S]SO 3 (2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states

  10. Functionality screen of streptavidin mutants by non-denaturing SDS-PAGE using biotin-4-fluorescein.

    Science.gov (United States)

    Humbert, Nicolas; Ward, Thomas R

    2008-01-01

    Site-directed mutagenesis or directed evolution of proteins often leads to the production of inactive mutants. For streptavidin and related proteins, mutations may lead to the loss of their biotin-binding properties. With high-throughput screening methodologies in mind, it is imperative to detect, prior to the high-density protein production, the bacteria that produce non-functional streptavidin isoforms. Based on the incorporation of biotin-4-fluorescein in streptavidin mutants present in Escherichia coli bacterial extracts, we detail a functional screen that allows the identification of biotin-binding streptavidin variants. Bacteria are cultivated in a small volume, followed by a rapid treatment of the cells; biotin-4-fluorescein is added to the bacterial extract and loaded on an Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) under non-denaturing conditions. Revealing is performed using a UV transilluminator. This screen is thus easy to implement, cheap and requires only readily available equipment.

  11. 27 CFR 19.464 - Denatured spirits inventories.

    Science.gov (United States)

    2010-04-01

    ... of Articles Inventories § 19.464 Denatured spirits inventories. Each proprietor shall take a physical inventory of all denatured spirits in the processing account at the close of each calendar quarter and at... inventories. 19.464 Section 19.464 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE...

  12. 27 CFR 20.144 - Packages of completely denatured alcohol.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Packages of completely denatured alcohol. 20.144 Section 20.144 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Sale...

  13. 27 CFR 20.261 - Records of completely denatured alcohol.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Records of completely denatured alcohol. 20.261 Section 20.261 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM...

  14. Development of two dimensional electrophoresis method using single chain DNA

    International Nuclear Information System (INIS)

    Ikeda, Junichi; Hidaka, So

    1998-01-01

    By combining a separation method due to molecular weight and a method to distinguish difference of mono-bases, it was aimed to develop a two dimensional single chain DNA labeled with Radioisotope (RI). From electrophoretic pattern difference of parent and variant strands, it was investigated to isolate the root module implantation control gene. At first, a Single Strand Conformation Polymorphism (SSCP) method using concentration gradient gel was investigated. As a result, it was formed that intervals between double chain and single chain DNAs expanded, but intervals of both single chain DNAs did not expand. On next, combination of non-modified acrylic amide electrophoresis method and Denaturing Gradient-Gel Electrophoresis (DGGE) method was examined. As a result, hybrid DNA developed by two dimensional electrophoresis arranged on two lines. But, among them a band of DNA modified by high concentration of urea could not be found. Therefore, in this fiscal year's experiments, no preferable result could be obtained. By the used method, it was thought to be impossible to detect the differences. (G.K.)

  15. Guanidinium-Induced Denaturation by Breaking of Salt Bridges.

    Science.gov (United States)

    Meuzelaar, Heleen; Panman, Matthijs R; Woutersen, Sander

    2015-12-07

    Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm(+) ) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm(+) can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm(+) -induced denaturation of a series of peptides containing Arg/Glu and Lys/Glu salt bridges that either stabilize or destabilize the folded conformation. The peptides containing stabilizing salt bridges are found to be denatured much more efficiently by Gdm(+) than the peptides containing destabilizing salt bridges. Complementary 2D-infrared measurements suggest a denaturation mechanism in which Gdm(+) binds to side-chain carboxylate groups involved in salt bridges. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Raw and renewable polymers

    CSIR Research Space (South Africa)

    Joseph, S

    2010-01-01

    Full Text Available in the permeability of the membrane and HO H3C H3C H2C H2C HO OH NH NH OH O OC C n O O O O Fig. 4 Structure of Chitin Raw and Renewable Polymers promoting internal osmotic imbalances. This results in leaching of electrolytes and proteins. 2... is often lost. In most cases this denaturation is not reversible. R-CH-COOH NH2 w Amino acid H2N COOHR a Amino acid Fig. 5 Structure of amino acid Raw and Renewable Polymers The solubilities of proteins vary considerably based on compositions...

  17. Thermal denaturation of A-DNA

    International Nuclear Information System (INIS)

    Valle-Orero, J; Wildes, A R; Theodorakopoulos, N; Cuesta-López, S; Peyrard, M; Garden, J-L; Danilkin, S

    2014-01-01

    The DNA molecule can take various conformational forms. Investigations focus mainly on the so-called ‘B-form’, schematically drawn in the famous paper by Watson and Crick [1]. This is the usual form of DNA in a biological environment and is the only form that is stable in an aqueous environment. Other forms, however, can teach us much about DNA. They have the same nucleotide base pairs for ‘building blocks’ as B-DNA, but with different relative positions, and studying these forms gives insight into the interactions between elements under conditions far from equilibrium in the B-form. Studying the thermal denaturation is particularly interesting because it provides a direct probe of those interactions which control the growth of the fluctuations when the ‘melting’ temperature is approached. Here we report such a study on the ‘A-form’ using calorimetry and neutron scattering. We show that it can be carried further than a similar study on B-DNA, requiring the improvement of thermodynamic models for DNA. (paper)

  18. Single-molecule denaturation mapping of DNA in nanofluidic channels

    DEFF Research Database (Denmark)

    Reisner, Walter; Larsen, Niels Bent; Silahtaroglu, Asli

    2010-01-01

    Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO (R)-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips...... and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence...

  19. Microwave-enhanced folding and denaturation of globular proteins

    DEFF Research Database (Denmark)

    Bohr, Henrik; Bohr, Jakob

    2000-01-01

    It is shown that microwave irradiation can affect the kinetics of the folding process of some globular proteins, especially beta-lactoglobulin. At low temperature the folding from the cold denatured phase of the protein is enhanced, while at a higher temperature the denaturation of the protein from...... its folded state is enhanced. In the latter case, a negative temperature gradient is needed for the denaturation process, suggesting that the effects of the microwaves are nonthermal. This supports the notion that coherent topological excitations can exist in proteins. The application of microwaves...

  20. Factors affecting the separation performance of proteins in capillary electrophoresis.

    Science.gov (United States)

    Zhu, Yueping; Li, Zhenqing; Wang, Ping; Shen, Lisong; Zhang, Dawei; Yamaguchi, Yoshinori

    2018-04-15

    Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. 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.

  2. Biomedical applications of capillary electrophoresis

    International Nuclear Information System (INIS)

    Kartsova, L A; Bessonova, E A

    2015-01-01

    The review deals with modern analytical approaches used in capillary electrophoresis for solving medical and biological problems: search for biomarkers of various diseases and rapid diagnosis based on characteristic profiles of biologically active compounds by capillary electrophoresis with mass spectrometric detection; monitoring of the residual drugs in biological fluids for evaluating the efficiency of drug therapy; testing of the enantiomeric purity of pharmaceutical products; the use of novel materials as components of stationary and pseudo-stationary phases in capillary electrophoresis and capillary electrochromatography to increase the selectivity of separation of components of complex matrices; and identification of various on-line preconcentration techniques to reduce the detection limits of biologically active analytes. A topical trend in capillary electrophoresis required in clinical practice, viz., the design of microfluidic systems, is discussed. The bibliography includes 173 references

  3. Light water reactors with a denatured thorium fuel cycle

    International Nuclear Information System (INIS)

    1978-05-01

    Discussed in this paper is the performance of denatured thorium fuel cycles in PWR plants of conventional design, such as those currently in operation or under construction. Although some improvement in U 3 O 8 utilization is anticipated in PWRs optimized explicitly for the denatured thorium fuel cycle, this paper is limited to a discussion of the performance of denatured thorium fuels in conventional PWRs and consequently the data presented is representative of the use of thorium fuel in existing PWRs or those presently under construction. In subsequent sections of this paper, the design of the PWR, its performance on the denatured thorium fuel cycle, safety, accident and environmental considerations, and technological status and R and D requirements are discussed

  4. Thermal denaturation of type I collagen vitrified gels

    International Nuclear Information System (INIS)

    Xia, Zhiyong; Calderon-Colon, Xiomara; Trexler, Morgana; Elisseeff, Jennifer; Guo, Qiongyu

    2012-01-01

    Highlights: ► We analyzed the denaturation of vitrigels synthesized under different conditions. ► Overall denaturation kinetics consisted of both reversible and irreversible steps. ► More stable vitrigels were formed under high level of vitrification. - Abstract: The denaturation kinetics of type I collagen vitrigels synthesized under different vitrification time and temperature were analyzed by the classical Kissinger approach and the advanced model free kinetics (AMFK) using the Vyazovkin algorithm. The AMFK successfully elucidated the overall denaturation into reversible and irreversible processes. Depending on vitrification conditions, the activation energy for the irreversible process ranged from 100 to 200 kJ/mol, and the reversible enthalpy ranged from 250 to 300 kJ/mol. All of these values increased with the vitrification time and temperature, indicating that a more stable and complex structure formed with increased vitrification. The classical Kissinger method predicted the presence of a critical temperate of approximately 60 °C for the transition between reversible and irreversible processes. Scanning electron microscopy revealed the presence of fibril structures in vitrigels both before and after full denaturation; however the fibrils had became thicker and rougher after denaturation.

  5. Serum protein fractionation using supported molecular matrix electrophoresis.

    Science.gov (United States)

    Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

    2013-08-01

    Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. "Cooperative collapse" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams.

    Science.gov (United States)

    Tischer, Alexander; Machha, Venkata R; Rösgen, Jörg; Auton, Matthew

    2018-02-19

    Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how ΔH and the urea m-value interconvert through the slope of c m versus T, (∂cm/∂T)=ΔH/(mT). This relationship permits the calculation of ΔH at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from ΔH obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of ΔH and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall

  7. Bioprocessing: Prospects for space electrophoresis

    Science.gov (United States)

    Bier, M.

    1977-01-01

    The basic principles of electrophoresis are reviewed in light of its past contributions to biology and medicine. The near-zero gravity environment of orbiting spacecraft may present some unique advantages for a variety of processes, by abolishing the major source of convection in fluids. As the ground-based development of electrophoresis was heavily influenced by the need to circumvent the effects of gravity, this process should be a prime candidate for space operation. Nevertheless, while a space facility for electrophoresis may overcome the limitations imposed by gravity, it will not necessarily overcome all problems inherent in electrophoresis. These are, mainly, electroosmosis and the dissipation of the heat generated by the electric field. The NASA program has already led to excellent coatings to prevent electroosmosis, while the need for heat dissipation will continue to impose limits on the actual size of equipment. It is also not excluded that, once the dominant force of gravity is eliminated, disturbances in fluid stability may originate from weaker forces, such as surface tension.

  8. Gel electrophoresis of inorganic cations

    International Nuclear Information System (INIS)

    Schoenhofer, F.; Grass, F.

    1978-01-01

    In order to be able to separate the largest possible amounts of substance, polyacryl amide gel (PAA) and silica gel are used as carrier for the electrophoresis. Milligramme quantities can easily be separated on PAA gel plates. Electrophoretic ion focussing considerably improves it. Separations of Sr/Y and lanthanoids were carried out. The behaviour of the readily soluble complexing agent acids on silica gel thin layers was minutely investigated and an interpretation of the focussing effect was derived. The conditions for separating radionuclides were optimized. A further improved separation can be achieved by a time sequence combination of normal electrophoresis and ion focussing. Selective isolation methods are advantageous to determine radionuclide traces in environmental samples. The selective adsorption on preformed deposits was transferred to electrophoresis. After pre-investigations on silica gel layers, strontium and barium could also be retained on PAA gel and radium on strontium sulphate in PAA, whereas the disturbing calcium can easily pass through. Cesium can also be retained by prussian blue in the electrophoresis. (orig.) [de

  9. Heavy water reactors on the denatured thorium cycles

    International Nuclear Information System (INIS)

    1978-05-01

    This paper presents preliminary technical and economic data to INFCE on the denatured U-233/Thorium fuel cycle for use in early comparisons of alternate nuclear systems. The once-through uranium fuel cycle is discussed in a companion paper. In presenting this preliminary information at this time, it is recognized that there are several other denatured thorium fuel cycles of potential interest, such as the U-235/thorium cycle which could be implemented at an earlier date. Information on these alternate cycles is currently being developed, and will be provided to INFCE when available

  10. On the radioimmunological determination of native and heat denaturated protein

    International Nuclear Information System (INIS)

    Menzel, E.J.; Glatz, F.; Technische Univ., Vienna

    1981-01-01

    Precipitation radioimmunoassay, solid phase radioimmunoassay and passive hemagglutination were examined for their efficiency in the determination of native or denaturated soy proteins. Native as well as autoclaved soy protein could be determined quantitatively in the precipitation radioimmunoassay, using antisera directed against the native product. In the solid phase technique only the autoclaved soy protein could be detected with high sensitivity. In the passive hemagglutination reaction, no agglutination could be observed with erythrocytes coated with autoclaved soy protein. Only antisera against the denaturated (autoclaved) soy protein agglutinated these erythrocytes. (orig.) [de

  11. Denaturation of membrane proteins and hyperthermic cell killing

    NARCIS (Netherlands)

    Burgman, Paulus Wilhelmus Johannes Jozef

    1993-01-01

    Summarizing: heat induced denaturation of membrane proteins is probably related to hyperthermic cell killing. Induced resistance of heat sensitive proteins seems to be involved in the development of thermotolerance. Although many questions remain still to be answered, it appears that HSP72, when

  12. Role of cyclobutane dimers in UV-denaturation of DNA

    International Nuclear Information System (INIS)

    Zavil'gel'skij, G.B.; Zuev, A.V.

    1978-01-01

    UV irradiation of double-stranded DNA produces local denatured regions. The evidence presented indicates that these single-stranded regions arise from photoproducts other than pyrimidine dimers. The irradiation of T2 DNA at 8x10 4 erg/mm 2 (254 nm) produces 6-8% thymine dimers, amd Tsub(mel) drops by 12-14 deg C, accompanied by a significant broadening of the transition profile. The kinetics of denatured region formation and lowering Tsub(mel) corresponds to that of formation of crosslinkages and differs markedly from the kinetics of formation of cyclobutane pyrimidine dimers. Treatment of UV-irradiated DNA with light in the presence of yeast photoreactivating enzyme monomerizes almost all thymine dimers but does not change the Tsub(mel). Local denatured regions are detected in UV-irradiated DNA and are absent from AcPhM-sensibilized DNA, which contains 20-25% thymine dimers, as determined by the accridine orange fluorescence technique. S1 nuclease from Aspergillis oryzae produces single-strand breaks in UV-irradiated DNA of phage PM2 but is not active on AcPhM-treated PM2 DNA, which contains about 50 thymine dimers. It is supposed that the formation of a cyclobutane dimer only weakens the hydrogen bonds in the AT base pair rather than breaks them. Local denatured regions are thought to arise from the accumulation in UV-irradiated DNA (254 nm) of the sufficient number of photoproducts with impaired ability to base pairing

  13. Cold denaturation of the HIV-1 protease monomer

    DEFF Research Database (Denmark)

    Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

    2017-01-01

    The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic approac...

  14. Western Blotting using Capillary Electrophoresis

    OpenAIRE

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

    2011-01-01

    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 a...

  15. Capillary electrophoresis systems and methods

    Science.gov (United States)

    Dorairaj, Rathissh [Hillsboro, OR; Keynton, Robert S [Louisville, KY; Roussel, Thomas J [Louisville, KY; Crain, Mark M [Georgetown, IN; Jackson, Douglas J [New Albany, IN; Walsh, Kevin M [Louisville, KY; Naber, John F [Goshen, KY; Baldwin, Richard P [Louisville, KY; Franco, Danielle B [Mount Washington, KY

    2011-08-02

    An embodiment of the invention is directed to a capillary electrophoresis apparatus comprising a plurality of separation micro-channels. A sample loading channel communicates with each of the plurality of separation channels. A driver circuit comprising a plurality of electrodes is configured to induce an electric field across each of the plurality of separation channels sufficient to cause analytes in the samples to migrate along each of the channels. The system further comprises a plurality of detectors configured to detect the analytes.

  16. 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

  17. Evaluation of the denaturing gradient gel electrophoresis-apparatus as a parameter influencing soil microbial community fingerprinting

    Czech Academy of Sciences Publication Activity Database

    Ascher, J.; Ceccherini, M.T.; Chroňáková, Alica; Jirout, Jiří; Borgogni, F.; Elhottová, Dana; Šimek, Miloslav; Pietramellara, G.

    2010-01-01

    Roč. 26, č. 9 (2010), s. 1721-1726 ISSN 0959-3993 R&D Projects: GA MŠk LC06066; GA AV ČR IAA600660605 Institutional research plan: CEZ:AV0Z60660521 Keywords : cattle impact * DGGE * DGGE interfering factors Subject RIV: EH - Ecology, Behaviour Impact factor: 1.214, year: 2010

  18. Association of Stremptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.

    Science.gov (United States)

    Abstract Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study, we used a cultureindependent met...

  19. Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil

    OpenAIRE

    Jousset, Alexandre; Lara, Enrique; Nikolausz, Marcell; Harms, Hauke; Chatzinotasa, Antonis

    2010-01-01

    Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to gre...

  20. Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Tan, H.

    1999-03-31

    The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

  1. New analytical portable instrument for microchip electrophoresis with electrochemical detection.

    Science.gov (United States)

    Fernández-la-Villa, Ana; Pozo-Ayuso, Diego F; Castaño-Alvarez, Mario

    2010-08-01

    A new portable instrument that includes a high voltage power supply, a bipotentiostat, and a chip holder has been especially developed for using microchips electrophoresis with electrochemical detection. The main unit of the instrument has dimensions of 150 x 165 x 70 mm (wxdxh) and consists of a four-outputs high voltage power supply with a maximum voltage of +/-3 KV and an acquisition system with two channels for dual amperometric (DC or pulsed amperometric detection) detection. Electrochemical detection has been selected as signal transduction method because it is relatively easily implemented, since nonoptical elements are required. The system uses a lithium-ion polymer battery and it is controlled from a desktop or laptop PC with a graphical user interface based on LabVIEW connected by serial RS232 or Bluetooth. The last part of the system consists of a reusable chip holder for housing the microchips, which contain all the electrical connections and reservoirs for making the work with microchips easy. The performance of the new instrument has been evaluated and compared with other commercially available apparatus using single- and dual-channel pyrex microchips for the separation of the neurotransmitters dopamine, epinephrine, and 3,4-dihydroxy-L-phenyl-alanine. The reduction of the size of the instrument has not affected the good performance of the separation and detection using microchips electrophoresis with electrochemical detection. Moreover, the new portable instrument paves the way for in situ analysis making the use of microchips electrophoresis easier.

  2. Polymer Coatings Reduce Electro-osmosis

    Science.gov (United States)

    Herren, Blair J.; Snyder, Robert; Shafer, Steven G.; Harris, J. Milton; Van Alstine, James M.

    1989-01-01

    Poly(ethylene glycol) film controls electrostatic potential. Electro-osmosis in quartz or glass chambers reduced or reversed by coating inside surface of chambers with monomacromolecular layers of poly(ethylene glycol). Stable over long times. Electrostatic potential across surface of untreated glass or plastic chamber used in electro-phoresis is negative and attracts cations in aqueous electrolyte. Cations solvated, entrains flow of electrolyte migrating toward cathode. Electro-osmotic flow interferes with desired electrophoresis of particles suspended in electrolyte. Polymer coats nontoxic, transparent, and neutral, advantageous for use in electrophoresis.

  3. Spectral shift controlled reactors, denatured U-233/thorium cycle

    International Nuclear Information System (INIS)

    1978-05-01

    This paper presents technical and economic data on the SSCR which may be of use in the International Fuel Cycle Evaluation Program to intercompare alternative nuclear systems. Included in this paper are data on the denatured U-233/thorium cycle. This cycle shows a proliferation advantage over more classical thorium fuel cycle (e.g., highly-enriched U-235/thorium or plutonium/thorium) due to the elimination of chemically-separable, concentrated fissile material from unirradiated nuclear fuel. The U-233 is denatured by mixing with depleted uranium to a concentration no greater than 12 w/o. An exogenous source of U-233 is assumed in this paper, since U-233 does not occur in nature and only a limited supply has been produced to date for research and development work

  4. Vibrational energy relaxation: proposed pathway of fast local chromatin denaturation

    International Nuclear Information System (INIS)

    Harder, D.; Greinert, R.

    2002-01-01

    The molecular mechanism responsible for the a component of exchange-type chromosome aberrations, of chromosome fragmentation and of reproductive cell death is one of the unsolved issues of radiation biology. Under review is whether vibrational energy relaxation in the constitutive biopolymers of chromatin, induced by inelastic energy deposition events and mediated via highly excited vibrational states, may provide a pathway of fast local chromatin denaturation, thereby producing the severe DNA lesion able to interact chemically with other, non-damaged chromatin. (author)

  5. Automation and integration of polymerase chain reaction with capillary electrophoresis for high throughput genotyping and disease diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, N.

    1999-02-12

    Genotyping is to detect specific loci in the human genome. These loci provide important information for forensic testing, construction of genetic linkage maps, gene related disease diagnosis and pharmacogenetic research. Genotyping is becoming more and more popular after these loci can be easily amplified by polymerase chain reaction (PCR). Capillary electrophoresis has its unique advantages for DNA analysis due to its fast heat dissipation and ease of automation. Four projects are described in which genotyping is performed by capillary electrophoresis emphasizing different aspects. First, the author demonstrates a principle to determine the genotype based on capillary electrophoresis system. VNTR polymorphism in the human D1S80 locus was studied. Second, the separation of four short tandem repeat (STR) loci vWF, THO1, TPOX and CSF1PO (CTTv) by using poly(ethylene oxide) (PEO) was studied in achieving high resolution and preventing rehybridization of the DNA fragments. Separation under denaturing, non-denaturing conditions and at elevated temperature was discussed. Third, a 250 {micro}m i.d., 365 {micro}m o.d. fused silica capillary was used as the microreactor for PCR. Fourth, direct PCR from blood was studied to simplify the sample preparation for genotyping to minimum.

  6. Agarose gel electrophoresis for the separation of DNA fragments.

    Science.gov (United States)

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-04-20

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate

  7. Preparation of denatured protein bone sterilized with gamma radiation

    International Nuclear Information System (INIS)

    Luna Z, D.

    2005-01-01

    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  8. 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.

  9. Polymer films

    Science.gov (United States)

    Granick, Steve; Sukhishvili, Svetlana A.

    2004-05-25

    A film contains a first polymer having a plurality of hydrogen bond donating moieties, and a second polymer having a plurality of hydrogen bond accepting moieties. The second polymer is hydrogen bonded to the first polymer.

  10. 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 ...

  11. Supramolecular gel electrophoresis of large DNA fragments.

    Science.gov (United States)

    Tazawa, Shohei; Kobayashi, Kazuhiro; Oyoshi, Takanori; Yamanaka, Masamichi

    2017-10-01

    Pulsed-field gel electrophoresis is a frequent technique used to separate exceptionally large DNA fragments. In a typical continuous field electrophoresis, it is challenging to separate DNA fragments larger than 20 kbp because they migrate at a comparable rate. To overcome this challenge, it is necessary to develop a novel matrix for the electrophoresis. Here, we describe the electrophoresis of large DNA fragments up to 166 kbp using a supramolecular gel matrix and a typical continuous field electrophoresis system. C 3 -symmetric tris-urea self-assembled into a supramolecular hydrogel in tris-boric acid-EDTA buffer, a typical buffer for DNA electrophoresis, and the supramolecular hydrogel was used as a matrix for electrophoresis to separate large DNA fragments. Three types of DNA marker, the λ-Hind III digest (2 to 23 kbp), Lambda DNA-Mono Cut Mix (10 to 49 kbp), and Marker 7 GT (10 to 165 kbp), were analyzed in this study. Large DNA fragments of greater than 100 kbp showed distinct mobility using a typical continuous field electrophoresis system. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. 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

  13. 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.

  14. Analytical biotechnology: Capillary electrophoresis and chromatography

    International Nuclear Information System (INIS)

    Horvath, C.; Nikelly, J.G.

    1990-01-01

    The papers describe the separation, characterization, and equipment required for the electrophoresis or chromatography of cyclic nucleotides, pharmaceuticals, therapeutic proteins, recombinant DNA products, pheromones, peptides, and other biological materials. One paper, On-column radioisotope detection for capillary electrophoresis, has been indexed separately for inclusion on the data base

  15. Membrane bridging and hemifusion by denaturated Munc18.

    Directory of Open Access Journals (Sweden)

    Yi Xu

    Full Text Available Neuronal Munc18-1 and members of the Sec1/Munc18 (SM protein family play a critical function(s in intracellular membrane fusion together with SNARE proteins, but the mechanism of action of SM proteins remains highly enigmatic. During experiments designed to address this question employing a 7-nitrobenz-2-oxa-1,3-diazole (NBD fluorescence de-quenching assay that is widely used to study lipid mixing between reconstituted proteoliposomes, we observed that Munc18-1 from squid (sMunc18-1 was able to increase the apparent NBD fluorescence emission intensity even in the absence of SNARE proteins. Fluorescence emission scans and dynamic light scattering experiments show that this phenomenon arises at least in part from increased light scattering due to sMunc18-1-induced liposome clustering. Nuclear magnetic resonance and circular dichroism data suggest that, although native sMunc18-1 does not bind significantly to lipids, sMunc18-1 denaturation at 37 °C leads to insertion into membranes. The liposome clustering activity of sMunc18-1 can thus be attributed to its ability to bridge two membranes upon (perhaps partial denaturation; correspondingly, this activity is hindered by addition of glycerol. Cryo-electron microscopy shows that liposome clusters induced by sMunc18-1 include extended interfaces where the bilayers of two liposomes come into very close proximity, and clear hemifusion diaphragms. Although the physiological relevance of our results is uncertain, they emphasize the necessity of complementing fluorescence de-quenching assays with alternative experiments in studies of membrane fusion, as well as the importance of considering the potential effects of protein denaturation. In addition, our data suggest a novel mechanism of membrane hemifusion induced by amphipathic macromolecules that does not involve formation of a stalk intermediate.

  16. Bioinspired phospholipid polymer biomaterials for making high performance artificial organs

    Directory of Open Access Journals (Sweden)

    K Ishihara

    2000-01-01

    Full Text Available Novel polymer biomaterials, which can be used in contact with blood, are prepared with strong inspiration from the surface structure of biomembrane. That is, the polymers with a phospholipid polar group in the side chain, 2-methacrylooyloxyethyl phosphorylcholine (MPC polymers were synthesized. The MPC polymers can inhibit surface-induced clot formation effectively, when they are in contact with blood even in the absence of an anticoagulant. This phenomenon was due to the reduction of plasma protein and suppression of denaturation of adsorbed proteins, that is the MPC polymers interact with blood components very mildly. As the molecular structure of the MPC polymer was easily designed by changing the monomer units and their composition, it could be applied to surface modification of artificial organs and biomedical devices for improving blood and tissue compatibility. Thus, the MPC polymers are useful polymer biomaterials for manufacturing high performance artificial organs and biomedical devices to provide safe medical treatments.

  17. Strategies for denaturing the weapons-grade plutonium stockpile

    International Nuclear Information System (INIS)

    Buckner, M.R.; Parks, P.B.

    1992-10-01

    In the next few years, approximately 50 metric tons of weapons-grade plutonium and 150 metric tons of highly-enriched uranium (HEU) may be removed from nuclear weapons in the US and declared excess. These materials represent a significant energy resource that could substantially contribute to our national energy requirements. HEU can be used as fuel in naval reactors, or diluted with depleted uranium for use as fuel in commercial reactors. This paper proposes to use the weapons-grade plutonium as fuel in light water reactors. The first such reactor would demonstrate the dual objectives of producing electrical power and denaturing the plutonium to prevent use in nuclear weapons

  18. Unfolding of hemoglobin variants--insights from urea gradient gel electrophoresis photon correlation spectroscopy and zeta potential measurements

    International Nuclear Information System (INIS)

    Bhattacharya, Jaydeep; GhoshMoulick, Ranjita; Choudhuri, Utpal; Chakrabarty, Prantar; Bhattacharya, Pranab K.; Lahiri, Prabir; Chakraborti, Bikas; Dasgupta, Anjan Kr.

    2004-01-01

    The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke's radii (obtained using the photon correlation spectroscopy) and zeta potential (measured using laser Doppler velocimetry) measurements were made at different denaturant concentrations. Addition of denaturant led to formation of extended structure, irrespective of the nature of the denaturant, as indicated by increase in Stoke's radii in both cases (urea and GdnHCl). The unexpected increase in electrophoretic mobility in case of urea could be explained in terms of a critical redistribution of negative charge at intermediate stages of the unfolding process. In case of GdnHCl, the higher ionic strength masked the charge effect. The mobility, being solely dependent on size, decreased at higher denaturant concentration. Incidentally, folding loci of other hemoglobin variants (e.g. HbE) or that of post-translationally modified hemoglobin (e.g. HbA1c) could be determined by studying the charge distribution and hydrodynamic radius at varying denaturing stress and in each case the gel migration profile could be approximately scaled by the ratio of charge and hydrodynamic diameter of the protein. While unfolding induced charge effect was most pronounced in HbA0 (and crystal ferrous hemoglobin), the unfolding induced aggregation (manifested by the increase in Stoke's radii) was predominantly observed in the variant forms HbE and HbA1c. Representing the proteins by a plot, in which charge and hydrodynamic diameter are on independent axes, may be a useful way of characterizing protein variants having similar migration profiles on native gels

  19. Unfolding of hemoglobin variants--insights from urea gradient gel electrophoresis photon correlation spectroscopy and zeta potential measurements

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Jaydeep; GhoshMoulick, Ranjita; Choudhuri, Utpal; Chakrabarty, Prantar; Bhattacharya, Pranab K.; Lahiri, Prabir; Chakraborti, Bikas; Dasgupta, Anjan Kr

    2004-09-27

    The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke's radii (obtained using the photon correlation spectroscopy) and zeta potential (measured using laser Doppler velocimetry) measurements were made at different denaturant concentrations. Addition of denaturant led to formation of extended structure, irrespective of the nature of the denaturant, as indicated by increase in Stoke's radii in both cases (urea and GdnHCl). The unexpected increase in electrophoretic mobility in case of urea could be explained in terms of a critical redistribution of negative charge at intermediate stages of the unfolding process. In case of GdnHCl, the higher ionic strength masked the charge effect. The mobility, being solely dependent on size, decreased at higher denaturant concentration. Incidentally, folding loci of other hemoglobin variants (e.g. HbE) or that of post-translationally modified hemoglobin (e.g. HbA1c) could be determined by studying the charge distribution and hydrodynamic radius at varying denaturing stress and in each case the gel migration profile could be approximately scaled by the ratio of charge and hydrodynamic diameter of the protein. While unfolding induced charge effect was most pronounced in HbA0 (and crystal ferrous hemoglobin), the unfolding induced aggregation (manifested by the increase in Stoke's radii) was predominantly observed in the variant forms HbE and HbA1c. Representing the proteins by a plot, in which charge and hydrodynamic diameter are on independent axes, may be a useful way of characterizing protein variants having similar migration profiles on

  20. Capillary electrophoresis and nanomaterials - Part I: Capillary electrophoresis of nanomaterials.

    Science.gov (United States)

    Adam, Vojtech; Vaculovicova, Marketa

    2017-10-01

    Nanomaterials are in analytical science used for a broad range of purposes, covering the area of sample pretreatment as well as separation, detection, and identification of target molecules. This part of the review covers capillary electrophoresis (CE) of nanomaterials and focuses on the application of CE as a method for characterization used during nanomaterial synthesis and modification as well as the monitoring of their properties and interactions with other molecules. The heterogeneity of the nanomaterial family is extremely large. Depending on different definitions of the term Nanomaterial/Nanoparticle, the group may cover metal and polymeric nanoparticles, carbon nanomaterials, liposomes and even dendrimers. Moreover, these nanomaterials are usually subjected to some kind of surface modification or functionalization, which broadens the diversity even more. Not only for purposes of verification of nanomaterial synthesis and batch-to-batch quality check, but also for determination the polydispersity and for functionality characterization on the nanoparticle surface, has CE offered very beneficial capabilities. Finally, the monitoring of interactions between nanomaterials and other (bio)molecules is easily performed by some kind of capillary electromigration technique. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. U.S. leans toward denatured thorium cycle

    International Nuclear Information System (INIS)

    Smock, R.

    1977-01-01

    Denatured thorium appears to be the most promising among the nonproliferating alternatives to the plutonium cycle, which the Carter Administration is trying to cancel. Criteria for a better system include uranium utilization comparable to current light water reactors and minimal separation of fissile material into the waste stream. Comparisons with other systems conclude that thorium is preferable because it can lead to an acceptable fast breeder. The thorium cycle can be placed in energy centers for sensitive facilities and can also be introduced into ongoing light water systems. Reprocessing can be handled in the centers, where thorium can be mixed with plutonium for use in reactors within the center, while light water reactors operate on the outside. Any fuel leaving the center would be unsuitable for weapons. Later adaptation to in-center fast breeders will extend energy supplies, although a thorium breeder will be less efficient than a plutonium fast breeder. Denatured thorium is a technical answer to a complex political problem, but those in the nuclear industry see the U.S. goal of a nonproliferating fuel as futile in the light of world politics and breeder efforts in other countries

  2. Cooperative unfolding of apolipoprotein A-1 induced by chemical denaturation.

    Science.gov (United States)

    Eckhardt, D; Li-Blatter, X; Schönfeld, H-J; Heerklotz, H; Seelig, J

    2018-05-25

    Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50-60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant K D and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Thermal denaturation of sunflower globulins in low moisture conditions

    International Nuclear Information System (INIS)

    Rouilly, A.; Orliac, O.; Silvestre, F.; Rigal, L.

    2003-01-01

    DSC analysis in pressure resisting pans of sunflower oil cake makes appear the endothermic peak of sunflower globulins denaturation. Its temperature decreases from 189.5 to 119.9 deg. C while the corresponding enthalpy increases from 2.6 to 3.3 J/g of sample, or from 6.7 to 12.2 J/g of dry protein, when the samples moisture content varies from 0 to 30.0% of the total weight. The plot of the denaturation temperature versus the moisture content is not linear but has a rounded global shape and seems to follow the hydration behavior of the proteins, modeled with the sorption isotherm. As it can be seen on scanning electron microscopy (SEM) micrographs, protein corpuscles 'melt' after such a thermal treatment and large aggregates form by coagulation. Moisture dependence of the 'fusion' temperature of native proteic organization, in low moisture conditions, offers so a new characterization method for the use of vegetable proteins in agro-materials

  4. Thermal denaturation of sunflower globulins in low moisture conditions

    Energy Technology Data Exchange (ETDEWEB)

    Rouilly, A.; Orliac, O.; Silvestre, F.; Rigal, L

    2003-03-05

    DSC analysis in pressure resisting pans of sunflower oil cake makes appear the endothermic peak of sunflower globulins denaturation. Its temperature decreases from 189.5 to 119.9 deg. C while the corresponding enthalpy increases from 2.6 to 3.3 J/g of sample, or from 6.7 to 12.2 J/g of dry protein, when the samples moisture content varies from 0 to 30.0% of the total weight. The plot of the denaturation temperature versus the moisture content is not linear but has a rounded global shape and seems to follow the hydration behavior of the proteins, modeled with the sorption isotherm. As it can be seen on scanning electron microscopy (SEM) micrographs, protein corpuscles 'melt' after such a thermal treatment and large aggregates form by coagulation. Moisture dependence of the 'fusion' temperature of native proteic organization, in low moisture conditions, offers so a new characterization method for the use of vegetable proteins in agro-materials.

  5. DNA electrophoresis through microlithographic arrays

    International Nuclear Information System (INIS)

    Sevick, E.M.; Williams, D.R.M.

    1996-01-01

    Electrophoresis is one of the most widely used techniques in biochemistry and genetics for size-separating charged molecular chains such as DNA or synthetic polyelectrolytes. The separation is achieved by driving the chains through a gel with an external electric field. As a result of the field and the obstacles that the medium provides, the chains have different mobilities and are physically separated after a given process time. The macroscopically observed mobility scales inversely with chain size: small molecules move through the medium quickly while larger molecules move more slowly. However, electrophoresis remains a tool that has yet to be optimised for most efficient size separation of polyelectrolytes, particularly large polyelectrolytes, e.g. DNA in excess of 30-50 kbp. Microlithographic arrays etched with an ordered pattern of obstacles provide an attractive alternative to gel media and provide wider avenues for size separation of polyelectrolytes and promote a better understanding of the separation process. Its advantages over gels are (1) the ordered array is durable and can be re-used, (2) the array morphology is ordered and can be standardized for specific separation, and (3) calibration with a marker polyelectrolyte is not required as the array is reproduced to high precision. Most importantly, the array geometry can be graduated along the chip so as to expand the size-dependent regime over larger chain lengths and postpone saturation. In order to predict the effect of obstacles upon the chain-length dependence in mobility and hence, size separation, we study the dynamics of single chains using theory and simulation. We present recent work describing: 1) the release kinetics of a single DNA molecule hooked around a point, frictionless obstacle and in both weak and strong field limits, 2) the mobility of a chain impinging upon point obstacles in an ordered array of obstacles, demonstrating the wide range of interactions possible between the chain and

  6. Application of denaturing high-performance liquid chromatography for monitoring sulfate-reducing bacteria in oil fields.

    Science.gov (United States)

    Priha, Outi; Nyyssönen, Mari; Bomberg, Malin; Laitila, Arja; Simell, Jaakko; Kapanen, Anu; Juvonen, Riikka

    2013-09-01

    Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H2S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (dsrB) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10(1) to 6 × 10(5) dsrB gene copies ml(-1). DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different dsrB compositions were detected at different geographical locations. The identified dsrB gene sequences belonged to several phylogenetic groups, such as Desulfovibrio, Desulfococcus, Desulfomicrobium, Desulfobulbus, Desulfotignum, Desulfonatronovibrio, and Desulfonauticus. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.

  7. Insight into the effect mechanism of urea-induced protein denaturation by dielectric spectroscopy.

    Science.gov (United States)

    Zhang, Cancan; Yang, Man; Zhao, Kongshuang

    2017-12-06

    Dielectric relaxation spectroscopy was applied to study how urea affects the phase transition of a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), which has been widely used as a protein model. It was found that there is a pronounced relaxation near 10 GHz for the ternary system of PNIPAM in urea aqueous solution. The temperature dependence of dielectric parameters indicates that urea can reduce the lower critical solution temperature (LCST) of PNIPAM, i.e., stabilize the globule state of PNIPAM and collapse the PNIPAM chains. Based on our results, the interaction mechanism of urea on the conformational transition of PNIPAM was presented: urea replaces water molecules directly bonding with PNIPAM and acts as the bridging agent for the adjacent side chains of PNIPAM. Accordingly, the mechanism with which urea denatures protein was deduced. In addition, it is worth mentioning that, from the temperature dependence of the dielectric parameters obtained in the presence of urea, an interesting phenomenon was found in which the effect of urea on PNIPAM seems to take 2 M as a unit. This result may be the reason why urea and TMAO exit marine fishes at a specific ratio of 2 : 1.

  8. 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.

  9. Denaturation/Renaturation of Organophosphorus Acid Anhydrolase (OPAA) Using Guanidinium Hydrochloride and Urea

    National Research Council Canada - National Science Library

    Ong, K. K; Sun, Z; Cheng, T. C; Wei, Y; Yuan, J. M; Yin, R

    2004-01-01

    .... Using organophosphorus acid anhydrolase (OPAA) as the model protein, a guanidinium hydrochloride and urea denaturation/renaturation study was conducted and measured both optically and enzymatically...

  10. Denaturation/Renaturation of Organophosphorus Acid Anhydrolase (OPAA) Using Guanidinium Hydrochloride and Urea

    National Research Council Canada - National Science Library

    Ong, K. K; Sun, Z; Cheng, T. C; Wei, Y; Yuan, J. M; Yin, R

    2004-01-01

    ...; thereby indicating conformational changes. Similar results were obtained with circular dichroism as the peak representing the alpha-helix conformation decreased as denaturant concentration was increased...

  11. Visualization of early events in acetic acid denaturation of HIV-1 protease: a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Aditi Narendra Borkar

    Full Text Available Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

  12. Recent progress in preparation and application of microfluidic chip electrophoresis

    International Nuclear Information System (INIS)

    Cong, Hailin; Xu, Xiaodan; Yu, Bing; Yuan, Hua; Peng, Qiaohong; Tian, Chao

    2015-01-01

    Since its discovery in 1990, microfluidic chip electrophoresis (MCE) has allowed the development of applications with small size, fast analysis, low cost, high integration density and automatic level, which are easy to carry and have made commercialization efficient. MCE has been widely used in the areas of environmental protection, biochemistry, medicine and health, clinical testing, judicial expertise, food sanitation, pharmaceutical checking, drug testing, agrochemistry, biomedical engineering and life science. As one of the foremost fields in the research of capillary electrophoresis, MCE is the ultimate frontier to develop the miniaturized, integrated, automated all-in-one instruments needed in modern analytical chemistry. By adopting the advanced technologies of micro-machining, lasers and microelectronics, and the latest research achievements in analytical chemistry and biochemistry, the sampling, separation and detection systems of commonly used capillary electrophoresis are integrated with high densities onto glass, quartz, silicon or polymer wafers to form the MCE, which can finish the analysis of multi-step operations such as injection, enrichment, reaction, derivatization, separation, and collection of samples in a portable, efficient and super high speed manner. With reference to the different technological achievements in this area, the latest developments in MCE are reviewed in this article. The preparation mechanisms, surface modifications, and properties of different materials in MCE are compared, and the different sampling, separation and detection systems in MCE are summarized. The performance of MCE in analysis of fluorescent substance, metallic ion, sugar, medicine, nucleic acid, DNA, amino acid, polypeptide and protein is discussed, and the future direction of development is forecast. (topical review)

  13. Recent progress in preparation and application of microfluidic chip electrophoresis

    Science.gov (United States)

    Cong, Hailin; Xu, Xiaodan; Yu, Bing; Yuan, Hua; Peng, Qiaohong; Tian, Chao

    2015-05-01

    Since its discovery in 1990, microfluidic chip electrophoresis (MCE) has allowed the development of applications with small size, fast analysis, low cost, high integration density and automatic level, which are easy to carry and have made commercialization efficient. MCE has been widely used in the areas of environmental protection, biochemistry, medicine and health, clinical testing, judicial expertise, food sanitation, pharmaceutical checking, drug testing, agrochemistry, biomedical engineering and life science. As one of the foremost fields in the research of capillary electrophoresis, MCE is the ultimate frontier to develop the miniaturized, integrated, automated all-in-one instruments needed in modern analytical chemistry. By adopting the advanced technologies of micro-machining, lasers and microelectronics, and the latest research achievements in analytical chemistry and biochemistry, the sampling, separation and detection systems of commonly used capillary electrophoresis are integrated with high densities onto glass, quartz, silicon or polymer wafers to form the MCE, which can finish the analysis of multi-step operations such as injection, enrichment, reaction, derivatization, separation, and collection of samples in a portable, efficient and super high speed manner. With reference to the different technological achievements in this area, the latest developments in MCE are reviewed in this article. The preparation mechanisms, surface modifications, and properties of different materials in MCE are compared, and the different sampling, separation and detection systems in MCE are summarized. The performance of MCE in analysis of fluorescent substance, metallic ion, sugar, medicine, nucleic acid, DNA, amino acid, polypeptide and protein is discussed, and the future direction of development is forecast.

  14. Characterization of asphaltenes by nonaqueous capillary electrophoresis

    NARCIS (Netherlands)

    Kok, W.T.; Tüdös, A.J.; Grutters, M.; Shepherd, A.G.

    2011-01-01

    Nonaqueous capillary electrophoresis was used for the separation and characterization of asphaltene samples from different sources. For the separation medium (background electrolyte), mixtures of tetrahydrofuran and a high-permittivity organic solvent could be used. The best results were obtained

  15. Irreversible denaturation of maltodextrin glucosidase studied by differential scanning calorimetry, circular dichroism, and turbidity measurements.

    Science.gov (United States)

    Goyal, Megha; Chaudhuri, Tapan K; Kuwajima, Kunihiro

    2014-01-01

    Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5-1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).

  16. Uranium production in thorium/denatured uranium fueled PWRs

    International Nuclear Information System (INIS)

    Arthur, W.B.

    1977-01-01

    Uranium-232 buildup in a thorium/denatured uranium fueled pressurized water reactor, PWR(Th), was studied using a modified version of the spectrum-dependent zero dimensional depletion code, LEOPARD. The generic Combustion Engineering System 80 reactor design was selected as the reactor model for the calculations. Reactors fueled with either enriched natural uranium and self-generated recycled uranium or uranium from a thorium breeder and self-generated recycled uranium were considered. For enriched natural uranium, concentrations of 232 U varied from about 135 ppM ( 232 U/U weight basis) in the zeroth generation to about 260 ppM ( 232 U/U weight basis) at the end of the fifth generation. For the case in which thorium breeder fuel (with its relatively high 232 U concentration) was used as reactor makeup fuel, concentrations of 232 U varied from 441 ppM ( 232 U/U weight basis) at discharge from the first generation to about 512 ppM ( 232 U/U weight basis) at the end of the fifth generation. Concentrations in freshly fabricated fuel for this later case were 20 to 35% higher than the discharge concentration. These concentrations are low when compared to those of other thorium fueled reactor types (HTGR and MSBR) because of the relatively high 238 U concentration added to the fuel as a denaturant. Excellent agreement was found between calculated and existing experimental values. Nevertheless, caution is urged in the use of these values because experimental results are very limited, and the relevant nuclear data, especially for 231 Pa and 232 U, are not of high quality

  17. Biodegradable Polymers

    OpenAIRE

    Vroman, Isabelle; Tighzert, Lan

    2009-01-01

    Biodegradable materials are used in packaging, agriculture, medicine and other areas. In recent years there has been an increase in interest in biodegradable polymers. Two classes of biodegradable polymers can be distinguished: synthetic or natural polymers. There are polymers produced from feedstocks derived either from petroleum resources (non renewable resources) or from biological resources (renewable resources). In general natural polymers offer fewer advantages than synthetic polymers. ...

  18. The generation of denatured reactor plutonium by different options of the fuel cycle

    Energy Technology Data Exchange (ETDEWEB)

    Broeders, C.H.M.; Kessler, G. [Inst. for Neutron Physics and Reactor Technology, Research Center Karlsruhe (Germany)

    2006-11-15

    Denatured (proliferation resistant) reactor plutonium can be generated in a number of different fuel cycle options. First denatured reactor plutonium can be obtained if, instead of low enriched U-235 PWR fuel, re-enriched U-235/U-236 from reprocessed uranium is used (fuel type A). Also the envisaged existing 2,500 t of reactor plutonium (being generated world wide up to the year 2010), mostly stored in intermediate fuel storage facilities at present, could be converted during a transition phase into denatured reactor plutonium by the options fuel type B and D. Denatured reactor plutonium could have the same safeguards standard as present low enriched (<20% U-235) LWR fuel. It could be incinerated by recycling once or twice in PWRs and subsequently by multi-recycling in FRs (CAPRA type or IFRs). Once denatured, such reactor plutonium could remain denatured during multiple recycling. In a PWR, e.g., denatured reactor plutonium could be destroyed at a rate of about 250 kg/GWey. While denatured reactor plutonium could be recycled and incinerated under relieved IAEA safeguards, neptunium would still have to be monitored by the IAEA in future for all cases in which considerable amounts of neptunium are produced. (orig.)

  19. Heat denaturation of soy glycinin : Influence of pH and ionic strength on molecular structure

    NARCIS (Netherlands)

    Lakemond, C.M.M.; Jongh, de H.H.J.; Hessing, M.; Gruppen, H.; Voragen, A.G.J.

    2000-01-01

    The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric

  20. 27 CFR 20.148 - Manufacture of articles with completely denatured alcohol.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Manufacture of articles with completely denatured alcohol. 20.148 Section 20.148 Alcohol, Tobacco Products and Firearms ALCOHOL... ALCOHOL AND RUM Sale and Use of Completely Denatured Alcohol § 20.148 Manufacture of articles with...

  1. Cation-Induced Stabilization and Denaturation of DNA Origami Nanostructures in Urea and Guanidinium Chloride.

    Science.gov (United States)

    Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

    2017-11-01

    The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and single-molecule biophysics. Here, the effect of Na + and Mg 2+ concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl. These inverse behaviors are rationalized by a salting-out of Gdm + to the hydrophobic DNA base stack. The effect of cation-induced DNA origami denaturation by GdmCl deserves consideration in the design of single-molecule studies and may potentially be exploited in future applications such as selective denaturation for purification purposes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Interim assessment of the denatured 233U fuel cycle: feasibility and nonproliferation characteristics

    International Nuclear Information System (INIS)

    Abbott, L.S.; Bartine, D.E.; Burns, T.J.

    1979-12-01

    A fuel cycle that employs 233 U denatured with 238 U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured 233 U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured 233 U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured 233 U fuel and are based on the energy center concept are evaluated

  3. 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

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

    NARCIS (Netherlands)

    Vrouwe, E.X.; Luttge, R.; Olthuis, W.; Berg, van den A.

    2005-01-01

    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

  5. Use of anionic denaturing detergents to purify insoluble proteins after overexpression

    Directory of Open Access Journals (Sweden)

    Schlager Benjamin

    2012-12-01

    Full Text Available Background Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. Results Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. Conclusion Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology.

  6. Polymer compound

    NARCIS (Netherlands)

    1995-01-01

    A Polymer compound comprising a polymer (a) that contains cyclic imidesgroups and a polymer (b) that contains monomer groups with a 2,4-diamino-1,3,5-triazine side group. According to the formula (see formula) whereby themole percentage ratio of the cyclic imides groups in the polymer compoundwith

  7. Polymer electronics

    CERN Document Server

    Hsin-Fei, Meng

    2013-01-01

    Polymer semiconductor is the only semiconductor that can be processed in solution. Electronics made by these flexible materials have many advantages such as large-area solution process, low cost, and high performance. Researchers and companies are increasingly dedicating time and money in polymer electronics. This book focuses on the fundamental materials and device physics of polymer electronics. It describes polymer light-emitting diodes, polymer field-effect transistors, organic vertical transistors, polymer solar cells, and many applications based on polymer electronics. The book also disc

  8. Preparative electrophoresis of industrial fission product solutions

    International Nuclear Information System (INIS)

    Tret, Joel

    1971-07-01

    The aim of this work is to contribute to the development of the continuous electrophoresis technique while studying its application in the preparative electrophoresis of industrial fission product solutions. The apparatus described is original. It was built for the purposes of the investigation and proved very reliable in operation. The experimental conditions necessary to maintain and supervise the apparatus in a state of equilibrium are examined in detail; their stability is an important factor, indispensable to the correct performance of an experiment. By subjecting an industrial solution of fission products to preparative electrophoresis it is possible, according to the experimental conditions, to prepare carrier-free radioelements of radiochemical purity (from 5 to 7 radioelements): 137 Cs, 90 Sr, 141+144 Ce, 91 Y, 95 Nb, 95 Zr, 103+106 Ru. (author) [fr

  9. Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

    International Nuclear Information System (INIS)

    Kresheck, G.C.; Erman, J.E.

    1988-01-01

    Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (t/sub m/) were 43.9 +- 1.4 and 63.3 +- 1.6 0 C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +- 1.3 0 C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase

  10. Renaturation of the androgen receptor after denaturation in SDS

    International Nuclear Information System (INIS)

    Johnson, M.P.; Young, C.Y.F.; Rowley, D.R.; Tindall, D.J.

    1986-01-01

    Renaturation of the steroid binding activity of receptor proteins is a potentially useful tool for their purification and analysis. Cytosol was prepared from rat Dunning prostate tumor in buffer containing molybdate and then denatured by addition of SDS buffer and heating. Aliquots were precipitated in cold acetone and the resulting pellets were washed and solubilized with a small volume of solution containing a chaotropic agent such as 6M guanidine, 8M urea, or 5M sodium iodide. After a 20-minute incubation, samples were diluted 20-fold with buffer containing 4nM [ 3 H]dihydrotestosterone with or without excess unlabeled dihydrotestosterone. Diluted samples were incubated at 0 0 C for varying periods of time prior to assay of bound radioactivity using hydroxylapatite. A time-course of renaturation after exposure to guanidine showed a steady increase of specific binding activity during the first 7 hrs post-dilution that remained stable up to 22 hrs. Experiments with guanidine consistently demonstrated that 25-50% of binding activity was recoverable. Preliminary results using urea or sodium iodide were similar. Efforts to optimize recovery and to characterize the renatured androgen receptor are in progress

  11. Thermally responsive silicon nanowire arrays for native/denatured-protein separation

    International Nuclear Information System (INIS)

    Wang Hongwei; Wang Yanwei; Yuan Lin; Wang Lei; Yang Weikang; Wu Zhaoqiang; Li Dan; Chen Hong

    2013-01-01

    We present our findings of the selective adsorption of native and denatured proteins onto thermally responsive, native-protein resistant poly(N-isopropylacrylamide) (PNIPAAm) decorated silicon nanowire arrays (SiNWAs). The PNIPAAm–SiNWAs surface, which shows very low levels of native-protein adsorption, favors the adsorption of denatured proteins. The amount of denatured-protein adsorption is higher at temperatures above the lower critical solution temperature (LCST) of PNIPAAm. Temperature cycling surrounding the LCST, which ensures against thermal denaturation of native proteins, further increases the amount of denatured-protein adsorption. Moreover, the PNIPAAm–SiNWAs surface is able to selectively adsorb denatured protein even from mixtures of different protein species; meanwhile, the amount of native proteins in solution is kept nearly at its original level. It is believed that these results will not only enrich current understanding of protein interactions with PNIPAAm-modified SiNWAs surfaces, but may also stimulate applications of PNIPAAm–SiNWAs surfaces for native/denatured protein separation. (paper)

  12. 27 CFR 19.41 - Claims on spirits, denatured spirits, articles, or wines lost or destroyed in bond.

    Science.gov (United States)

    2010-04-01

    ..., denatured spirits, articles, or wines lost or destroyed in bond. 19.41 Section 19.41 Alcohol, Tobacco... DISTILLED SPIRITS PLANTS Taxes Claims § 19.41 Claims on spirits, denatured spirits, articles, or wines lost..., relating to the destruction or loss of spirits, denatured spirits, articles, or wines in bond, shall be...

  13. Undergraduate physics laboratory: Electrophoresis in chromatography paper

    Science.gov (United States)

    Hyde, Alexander; Batishchev, Oleg

    2015-12-01

    An experiment studying the physical principles of electrophoresis in liquids was developed for an undergraduate laboratory. We have improved upon the standard agarose gel electrophoresis experimental regime with a straightforward and cost-effective procedure, in which drops of widely available black food coloring were separated by electric field into their dye components on strips of chromatography paper soaked in a baking soda/water solution. Terminal velocities of seven student-safe dyes were measured as a function of the electric potential applied along the strips. The molecular mobility was introduced and calculated by analyzing data for a single dye. Sources of systematic and random errors were investigated.

  14. Urea-temperature phase diagrams capture the thermodynamics of denatured state expansion that accompany protein unfolding

    Science.gov (United States)

    Tischer, Alexander; Auton, Matthew

    2013-01-01

    We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea-induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two-state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m-value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea-denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions. PMID:23813497

  15. 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.

  16. Protein denaturation and functional properties of Lenient Steam Injection heat treated whey protein concentrate

    DEFF Research Database (Denmark)

    Dickow, Jonatan Ahrens; Kaufmann, Niels; Wiking, Lars

    2012-01-01

    Whey protein concentrate (WPC) was heat treated by use of the novel heat treatment method of Lenient Steam Injection (LSI) to elucidate new functional properties in relation to heat-induced gelation of heat treated WPC. Denaturation was measured by both DSC and FPLC, and the results of the two...... methods were highly correlated. Temperatures of up to 90 °C were applicable using LSI, whereas only 68 °C could be reached by plate heat exchange before coagulation/fouling. Denaturation of whey proteins increased with increasing heat treatment temperature up to a degree of 30–35% denaturation at 90 °C...

  17. 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 prototype-test cycle - significantly faster than mold based fabrication techniques - we optimize the fsPAG architecture to minimize injection dispersion for rapid (prototyping of the fsPAGE provides researchers a powerful tool for developing custom analytical assays. We highlight the utility of assay customization by fabricating a polyacrylamide gel with a spatial pore-size distribution and demonstrate the resulting enhancement in separation performance over a uniform gel. Further, we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAG array layout matches that of a 96-well plate to facilitate integration of the planar free standing gel array with multi-channel pipettes while remaining compatible with conventional slab-gel PAGE reagents, such as staining for label-free protein detection. Notably, the entire fsPAGE workflow from fabrication, to operation, and readout uses readily available materials and instruments - making this technique highly accessible.

  18. 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 ...

  19. Contemporary sample stacking in analytical electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Šlampová, Andrea; Malá, Zdeňka; Pantůčková, Pavla; Gebauer, Petr; Boček, Petr

    2013-01-01

    Roč. 34, č. 1 (2013), s. 3-18 ISSN 0173-0835 R&D Projects: GA ČR GAP206/10/1219 Institutional support: RVO:68081715 Keywords : biological samples * stacking * trace analysis * zone electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.161, year: 2013

  20. Electrophoresis test prevalence, requesting patterns, yield and ...

    African Journals Online (AJOL)

    Most of the appropriate SPE test requests were from clinical haematology, renal ... implementation of principles of demand management and the ... electrophoresis (IFE)) in a South African (SA) pathology laboratory setting are limited. Objectives. ... (NHLS) hospital information system database from 1 July 2010 to. 30 June ...

  1. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  2. Contemporary sample stacking in analytical electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Malá, Zdeňka; Šlampová, Andrea; Křivánková, Ludmila; Gebauer, Petr; Boček, Petr

    2015-01-01

    Roč. 36, č. 1 (2015), s. 15-35 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA13-05762S Institutional support: RVO:68081715 Keywords : biological samples * stacking * trace analysis * zone electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.482, year: 2015

  3. Matching Two-dimensional Gel Electrophoresis' Spots

    DEFF Research Database (Denmark)

    Dos Anjos, António; AL-Tam, Faroq; Shahbazkia, Hamid Reza

    2012-01-01

    This paper describes an approach for matching Two-Dimensional Electrophoresis (2-DE) gels' spots, involving the use of image registration. The number of false positive matches produced by the proposed approach is small, when compared to academic and commercial state-of-the-art approaches. This ar...

  4. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...

  5. Concentration polarization in nanochannel DNA electrophoresis

    NARCIS (Netherlands)

    Dubsky, P.; Das, Siddhartha; van den Berg, Albert; Eijkel, Jan C.T.

    2011-01-01

    We demonstrate that the large field electrophoresis of a single DNA molecule in nanofluidic systems is accompanied by concentration polarization. We illustrate this phenomena by utilizing our electrophoretic simulation tool SIMUL. First we in-vestigate a simple system with univalent strong

  6. Sodium dodecyl sulfate-capillary gel electrophoresis of polyethylene glycolylated interferon alpha.

    Science.gov (United States)

    Na, Dong H; Park, Eun J; Youn, Yu S; Moon, Byung W; Jo, Yeong W; Lee, Sung H; Kim, Won-Bae; Sohn, Yeowon; Lee, Kang C

    2004-02-01

    Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.

  7. Studying pressure denaturation of a protein by molecular dynamics simulations.

    Science.gov (United States)

    Sarupria, Sapna; Ghosh, Tuhin; García, Angel E; Garde, Shekhar

    2010-05-15

    Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This "unfolding-up-on-squeezing" is counter-intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results-that pressure denatured states are water-swollen, and theoretical results-that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states-their water-swollen nature, retention of secondary structure, and overall compactness-mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure-dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately -60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500-2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water-swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties

  8. Sample Stacking in capillary zone electrophoresis : Principles, advantages and limitations

    NARCIS (Netherlands)

    Beckers, J.L.; Bocek, P.

    2000-01-01

    The principles of stacking procedures are described and their properties are discussed, including the fundamentals of the behavior of zone boundaries and the consequences of the self-correcting properties of boundaries in moving boundary electrophoresis, isotachophoresis, and zone electrophoresis.

  9. 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)

  10. How Chain Length and Charge Affect Surfactant Denaturation of Acyl Coenzyme A Binding Protein (ACBP)

    DEFF Research Database (Denmark)

    Andersen, Kell Kleiner; Otzen, Daniel

    2009-01-01

    maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle......Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl...... constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex...

  11. On the Use of Potential Denaturing Agents for Ethanol in Direct Ethanol Fuel Cells

    Directory of Open Access Journals (Sweden)

    Domnik Bayer

    2011-01-01

    Full Text Available Acidic or alkaline direct ethanol fuel cells (DEFCs can be a sustainable alternative for power generation if they are fuelled with bio-ethanol. However, in order to keep the fuel cheap, ethanol has to be exempted from tax on spirits by denaturing. In this investigation the potential denaturing agents fusel oil, tert-butyl ethyl ether, and Bitrex were tested with regard to their compatibility with fuel cells. Experiments were carried out both in sulphuric acid and potassium hydroxide solution. Beside, basic electrochemical tests, differential electrochemical mass spectrometry (DEMS and fuel cell tests were conducted. It was found that fusel oil is not suitable as denaturing agent for DEFC. However, tert-butyl ethyl ether does not seem to hinder the ethanol conversion as much. Finally, a mixture of tert-butyl ethyl ether and Bitrex can be proposed as promising candidate as denaturing agent for use in acidic and alkaline DEFC.

  12. Nonradioactive telomerase activity assay by microchip electrophoresis: privileges to the classical gel electrophoresis assay.

    Science.gov (United States)

    Zhelev, Zhivko; Bakalova, Rumiana; Ewis, Ashraf; Ohba, Hideki; Ishikawa, Mitsuru; Baba, Yoshinobu

    2005-08-01

    The present study accents on the privileges of microchip-based electrophoresis to the conventional gel electrophoresis in separation of telomerase repeat amplification protocol/polymerase chain reaction (PCR) ladder products obtained in telomerase-catalyzed reaction in cancer cells. We try to clarify the interpretation of the results obtained by both electrophoretic procedures and to avoid misinterpretation as a result of PCR-dependent artefacts.

  13. The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs.

    Science.gov (United States)

    Liang, Pengfei; Jiang, Bimei; Lv, Chunliu; Huang, Xu; Sun, Li; Zhang, Pihong; Huang, Xiaoyuan

    2013-10-01

    The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burn model in Sprague-Dawley rats and the heat denatured cell model (52°C, 35s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin. Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs. The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF. We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. On the Use of Potential Denaturing Agents for Ethanol in Direct Ethanol Fuel Cells

    OpenAIRE

    Domnik Bayer; Florina Jung; Birgit Kintzel; Martin Joos; Carsten Cremers; Dierk Martin; Jörg Bernard; Jens Tübke

    2011-01-01

    Acidic or alkaline direct ethanol fuel cells (DEFCs) can be a sustainable alternative for power generation if they are fuelled with bio-ethanol. However, in order to keep the fuel cheap, ethanol has to be exempted from tax on spirits by denaturing. In this investigation the potential denaturing agents fusel oil, tert-butyl ethyl ether, and Bitrex were tested with regard to their compatibility with fuel cells. Experiments were carried out both in sulphuric acid and potassium hydroxide solution...

  15. PHOTOREFRACTIVE POLYMERS

    NARCIS (Netherlands)

    Morichere, D; Malliaras, G.G; Krasnikov, V.V.; Bolink, H.J; Hadziioannou, G

    The use of polymers as photorefractive materials offers many advantages : flexibility in synthesis, doping, processing and low cost. The required functionalities responsible for photorefractivity, namely charge generation, transport, trapping and linear electrooptic effect are given in the polymer

  16. Photorefractive polymers

    NARCIS (Netherlands)

    Bolink, Hendrik Jan; Hadziioannou, G

    1997-01-01

    This thesis describes the synthesis and properties of photorefractive polymers. Photorefractive polymers are materials in which the refractive index can be varied by the interaction with light. Unlike in numerous other photosensitive materials, in photorefractive materials this occurs via

  17. Interim assessment of the denatured 233U fuel cycle: feasibility and nonproliferation characteristics

    International Nuclear Information System (INIS)

    Abbott, L.S.; Bartine, D.E.; Burns, T.J.

    1978-12-01

    A fuel cycle that employs 233 U denatured with 238 U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured 233 U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured 233 U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured 233 U fuel and are based on the energy center concept are evaluated. Under this concept, dispersed power reactors fueled with denatured or low-enriched uranium fuel are supported by secure energy centers in which sensitive activities of the nuclear cycle are performed. These activities include 233 U production by Pu-fueled transmuters (thermal or fast reactors) and reprocessing. A summary chapter presents the most significant conclusions from the study and recommends areas for future work

  18. Interim assessment of the denatured /sup 233/U fuel cycle: feasibility and nonproliferation characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Abbott, L.S.; Bartine, D.E.; Burns, T.J. (eds.)

    1978-12-01

    A fuel cycle that employs /sup 233/U denatured with /sup 238/U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured /sup 233/U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured /sup 233/U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured /sup 233/U fuel and are based on the energy center concept are evaluated. Under this concept, dispersed power reactors fueled with denatured or low-enriched uranium fuel are supported by secure energy centers in which sensitive activities of the nuclear cycle are performed. These activities include /sup 233/U production by Pu-fueled transmuters (thermal or fast reactors) and reprocessing. A summary chapter presents the most significant conclusions from the study and recommends areas for future work.

  19. Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

    Science.gov (United States)

    Wei, Haiyan; Yang, Lijiang; Gao, Yi Qin

    2010-09-16

    Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of β-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

  20. 21 CFR 862.2485 - Electrophoresis apparatus for clinical use.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Electrophoresis apparatus for clinical use. 862.2485 Section 862.2485 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Instruments § 862.2485 Electrophoresis apparatus for clinical use. (a) Identification. An electrophoresis...

  1. Polymer Brushes

    NARCIS (Netherlands)

    Vos, de W.M.; Kleijn, J.M.; Keizer, de A.; Cosgrove, T.; Cohen Stuart, M.A.

    2010-01-01

    A polymer brush can be defined as a dense array of polymers end-attached to an interface that stretch out into the surrounding medium. Polymer brushes have been investigated for the past 30 years and have shown to be an extremely useful tool to control interfacial properties. This review is intended

  2. Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands

    DEFF Research Database (Denmark)

    Pedersen, Katrine E; Einholm, Anja P; Christensen, Anni

    2003-01-01

    Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As com...

  3. Microfluidic chip-capillary electrophoresis devices

    CERN Document Server

    Fung, Ying Sing; Du, Fuying; Guo, Wenpeng; Ma, Tongmei; Nie, Zhou; Sun, Hui; Wu, Ruige; Zhao, Wenfeng

    2015-01-01

    Capillary electrophoresis (CE) and microfluidic chip (MC) devices are relatively mature technologies, but this book demonstrates how they can be integrated into a single, revolutionary device that can provide on-site analysis of samples when laboratory services are unavailable. By introducing the combination of CE and MC technology, Microfluidic Chip-Capillary Electrophoresis Devices broadens the scope of chemical analysis, particularly in the biomedical, food, and environmental sciences. The book gives an overview of the development of MC and CE technology as well as technology that now allows for the fabrication of MC-CE devices. It describes the operating principles that make integration possible and illustrates some achievements already made by the application of MC-CE devices in hospitals, clinics, food safety, and environmental research. The authors envision further applications for private and public use once the proof-of-concept stage has been passed and obstacles to increased commercialization are ad...

  4. 27 CFR 19.32 - Assessment of tax on spirits, denatured spirits, or wines in bond which are lost, destroyed or...

    Science.gov (United States)

    2010-04-01

    ... spirits, denatured spirits, or wines in bond which are lost, destroyed or removed without authorization... spirits, denatured spirits, or wines in bond which are lost, destroyed or removed without authorization. When spirits, denatured spirits, or wines in bond are lost or destroyed (except spirits, denatured...

  5. Optically controlled electrophoresis with a photoconductive substrate

    Science.gov (United States)

    Inami, Wataru; Nagashima, Taiki; Kawata, Yoshimasa

    2018-05-01

    A photoconductive substrate is used to perform electrophoresis. Light-induced micro-particle flow manipulation is demonstrated without using a fabricated flow channel. The path along which the particles were moved was formed by an illuminated light pattern on the substrate. Because the substrate conductivity and electric field distribution can be modified by light illumination, the forces acting on the particles can be controlled. This technique has potential applications as a high functionality analytical device.

  6. Detection of telomerase activity using microchip electrophoresis.

    Science.gov (United States)

    Karasawa, Koji; Arakawa, Hidetoshi

    2015-07-01

    Telomerase participates in malignant transformation or immortalization of cells and thus has attracted attention as an anticancer drug target and diagnostic tumor marker. The telomeric repeat amplification protocol (TRAP) and improved TRAP methods (TRAP-fluorescence, TRAP-hybridization, etc.) are widely used forms of this telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplification of telomeric repeats by polymerase chain reaction (PCR), making these TRAP methods time consuming and technically demanding. In this study we developed a novel telomerase assay using microchip electrophoresis for rapid and highly sensitive detection of telomerase activity in cancer cells. The mixed gel of 0.8% hydroxypropyl methylcellulose (HPMC) and 0.3% polyethylene oxide (PEO) with SYBR Gold (fluorescent reagent) was used for microchip electrophoresis. As a result, the product amplified by a telomerase-positive cell could be measured in one cell per assay and detected with high reproducibility (CV=0.67%) in the short time of 100s. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.

    Science.gov (United States)

    Melani, Rafael D; Skinner, Owen S; Fornelli, Luca; Domont, Gilberto B; Compton, Philip D; Kelleher, Neil L

    2016-07-01

    Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric l-amino acid oxidase (∼130 kDa) and the multichain toxin cobra venom factor (∼147 kDa). The l-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length.

    Science.gov (United States)

    Kobayashi, Shinya; Fujii, Sotaro; Koga, Aya; Wakai, Satoshi; Matubayasi, Nobuyuki; Sambongi, Yoshihiro

    2017-07-01

    Reversible denaturation of Pseudomonas aeruginosa cytochrome c 551 (PAc 551 ) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc 551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc 551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc 551 , for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.

  9. Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

    Directory of Open Access Journals (Sweden)

    M.R. Hespanhol

    2002-03-01

    Full Text Available Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18 and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA by mouse peritoneal macrophages. We observed that a macrophages are able to recognize (bind to these red cells, b this interaction can be inhibited by denatured BSA in the fluid phase, c there is no phagocytosis of these particles by normal macrophages, d phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A.

  10. Alcohol-induced structural transitions in the acid-denatured Bacillus licheniformis α-amylase

    Directory of Open Access Journals (Sweden)

    Adyani Azizah Abd Halim

    2017-01-01

    Full Text Available Alcohol-induced structural changes in the acid-denatured Bacillus licheniformis α-amylase (BLA at pH 2.0 were studied by far-ultra violet circular dichroism, intrinsic, three-dimensional and 8-anilino-1-naphthalene sulfonic acid (ANS fluorescence, acrylamide quenching and thermal denaturation. All the alcohols used in this study produced partial refolding in the acid-denatured BLA as evident from the increased mean residue ellipticity at 222 nm, increased intrinsic fluorescence and decreased ANS fluorescence. The order of effectiveness of these alcohols to induce a partially folded state of BLA was found to be: 2,2,2-trifluoroethanol/tert-butanol > 1-propanol/2-propanol > 2-chloroethanol > ethanol > methanol. Three-dimensional fluorescence and acrylamide quenching results obtained in the presence of 5.5 M tert-butanol also suggested formation of a partially folded state in the acid-denatured BLA. However, 5.5 M tert-butanol-induced state of BLA showed a non-cooperative thermal transition. All these results suggested formation of a partially folded state of the acid-denatured BLA in the presence of these alcohols. Furthermore, their effectiveness was found to be guided by their chain length, position of methyl groups and presence of the substituents.

  11. Drying and denaturation characteristics of whey protein isolate in the presence of lactose and trehalose.

    Science.gov (United States)

    Haque, M Amdadul; Chen, Jie; Aldred, Peter; Adhikari, Benu

    2015-06-15

    The denaturation kinetics of whey protein isolate (WPI), in the presence and absence of lactose and trehalose, was quantified in a convective air-drying environment. Single droplets of WPI, WPI-lactose and WPI-trehalose were dried in conditioned air (2.5% RH, 0.5m/s air velocity) at two temperatures (65°C and 80°C) for 500s. The initial solid concentration of these solutions was 10% (w/v) in all the samples. Approximately 68% of WPI was denatured when it was dried in the absence of sugars. Addition of 20% trehalose prevented the irreversible denaturation of WPI at both temperatures. Thirty percent lactose was required to prevent denaturation of WPI at 65°C and the same amount of lactose protected only 70% of WPI from denaturation at 80°C. The secondary structures of WPI were found to be altered by the drying-induced stresses, even in the presence of 20% trehalose and 30% lactose. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Neutronics calculations for denatured molten salt reactors: Assessing resource requirements and proliferation-risk attributes

    International Nuclear Information System (INIS)

    Ahmad, Ali; McClamrock, Edward B.; Glaser, Alexander

    2015-01-01

    Highlights: • We study the proliferation-risk and resource attributes of denatured MSRs. • MSRs offer significantly better resource efficiency compared to light-water reactors. • Denatured single-fluid MSRs reactors offer promising non-proliferation attributes. - Abstract: Molten salt reactors (MSRs) are often advocated as a radical but worthwhile alternative to traditional reactor concepts based on solid fuels. This article builds upon the existing research into MSRs to model and simulate the operation of thorium-fueled single-fluid and two-fluid reactors. The analysis is based on neutronics calculations and focuses on denatured MSR systems. Resource utilization and basic proliferation-risk attributes are compared to those of standard light-water reactors. Depending on specific design choices, even fully denatured reactors could reduce uranium and enrichment requirements by a factor of 3–4. Overall, denatured single-fluid designs appear as the most promising candidate technology minimizing both design complexity and overall proliferation risks despite being somewhat less attractive from the perspective of resource utilization

  13. Importance of core electrostatic properties on the electrophoresis of a soft particle

    Science.gov (United States)

    De, Simanta; Bhattacharyya, Somnath; Gopmandal, Partha P.

    2016-08-01

    The impact of the volumetric charged density of the dielectric rigid core on the electrophoresis of a soft particle is analyzed numerically. The volume charge density of the inner core of a soft particle can arise for a dendrimer structure or bacteriophage MS2. We consider the electrokinetic model based on the conservation principles, thus no conditions for Debye length or applied electric field is imposed. The fluid flow equations are coupled with the ion transport equations and the equation for the electric field. The occurrence of the induced nonuniform surface charge density on the outer surface of the inner core leads to a situation different from the existing analysis of a soft particle electrophoresis. The impact of this induced surface charge density together with the double-layer polarization and relaxation due to ion convection and electromigration is analyzed. The dielectric permittivity and the charge density of the core have a significant impact on the particle electrophoresis when the Debye length is in the order of the particle size. We find that by varying the ionic concentration of the electrolyte, the particle can exhibit reversal in its electrophoretic velocity. The role of the polymer layer softness parameter is addressed in the present analysis.

  14. Radiation-induced DNA breaks detected by immuno labelling of poly(ADP-ribose) in CHO cells. Standardization by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Varlet, P.; Bidon, N.; Noel, G.; Averbeck, D.; Salamero, J.; DeMurcia, G.

    1998-01-01

    The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos)ylation to follow and estimate γ-ray-induced DNA fragmentation at the level of isolated cells after γ-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immuno-labelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after γ-irradiation. (authors)

  15. Polymer Electrolytes

    Science.gov (United States)

    Hallinan, Daniel T.; Balsara, Nitash P.

    2013-07-01

    This review article covers applications in which polymer electrolytes are used: lithium batteries, fuel cells, and water desalination. The ideas of electrochemical potential, salt activity, and ion transport are presented in the context of these applications. Potential is defined, and we show how a cell potential measurement can be used to ascertain salt activity. The transport parameters needed to fully specify a binary electrolyte (salt + solvent) are presented. We define five fundamentally different types of homogeneous electrolytes: type I (classical liquid electrolytes), type II (gel electrolytes), type III (dry polymer electrolytes), type IV (dry single-ion-conducting polymer electrolytes), and type V (solvated single-ion-conducting polymer electrolytes). Typical values of transport parameters are provided for all types of electrolytes. Comparison among the values provides insight into the transport mechanisms occurring in polymer electrolytes. It is desirable to decouple the mechanical properties of polymer electrolyte membranes from the ionic conductivity. One way to accomplish this is through the development of microphase-separated polymers, wherein one of the microphases conducts ions while the other enhances the mechanical rigidity of the heterogeneous polymer electrolyte. We cover all three types of conducting polymer electrolyte phases (types III, IV, and V). We present a simple framework that relates the transport parameters of heterogeneous electrolytes to homogeneous analogs. We conclude by discussing electrochemical stability of electrolytes and the effects of water contamination because of their relevance to applications such as lithium ion batteries.

  16. Star Polymers.

    Science.gov (United States)

    Ren, Jing M; McKenzie, Thomas G; Fu, Qiang; Wong, Edgar H H; Xu, Jiangtao; An, Zesheng; Shanmugam, Sivaprakash; Davis, Thomas P; Boyer, Cyrille; Qiao, Greg G

    2016-06-22

    Recent advances in controlled/living polymerization techniques and highly efficient coupling chemistries have enabled the facile synthesis of complex polymer architectures with controlled dimensions and functionality. As an example, star polymers consist of many linear polymers fused at a central point with a large number of chain end functionalities. Owing to this exclusive structure, star polymers exhibit some remarkable characteristics and properties unattainable by simple linear polymers. Hence, they constitute a unique class of technologically important nanomaterials that have been utilized or are currently under audition for many applications in life sciences and nanotechnologies. This article first provides a comprehensive summary of synthetic strategies towards star polymers, then reviews the latest developments in the synthesis and characterization methods of star macromolecules, and lastly outlines emerging applications and current commercial use of star-shaped polymers. The aim of this work is to promote star polymer research, generate new avenues of scientific investigation, and provide contemporary perspectives on chemical innovation that may expedite the commercialization of new star nanomaterials. We envision in the not-too-distant future star polymers will play an increasingly important role in materials science and nanotechnology in both academic and industrial settings.

  17. Polymer chemistry (revised edition)

    International Nuclear Information System (INIS)

    Kim, Jae Mum

    1987-02-01

    This book deals with polymer chemistry, which is divided into fourteen chapters. The contents of this book are development of polymer chemistry, conception of polymer, measurement of polymer chemistry, conception of polymer, measurement of polymer, molecule structure of polymer, thermal prosperities of solid polymer, basic theory of polymerization, radical polymerization, ion polymerization, radical polymerization, copolymerization, polymerization by step-reaction, polymer reaction, crown polymer and inorganic polymer on classification and process of creation such as polymeric sulfur and carbon fiber.

  18. Nonsurgical Transurethral Radiofrequency Collagen Denaturation: Results at Three Years after Treatment

    Directory of Open Access Journals (Sweden)

    Denise M. Elser

    2011-01-01

    Full Text Available Objective. To assess treatment efficacy and quality of life in women with stress urinary incontinence 3 years after treatment with nonsurgical transurethral radiofrequency collagen denaturation. Methods. This prospective study included 139 women with stress urinary incontinence due to bladder outlet hypermobility. Radiofrequency collagen denaturation was performed using local anesthesia in an office setting. Assessments included incontinence quality of life (I-QOL and urogenital distress inventory (UDI-6 instruments. Results. In total, 139 women were enrolled and 136 women were treated (mean age, 47 years. At 36 months, intent-to-treat analysis (n=139 revealed significant improvements in quality of life. Mean I-QOL score improved 17 points from baseline (P=.0004, while mean UDI-6 score improved (decreased 19 points (P=.0005. Conclusions. Transurethral collagen denaturation is a low-risk, office-based procedure that results in durable quality-of-life improvements in a significant proportion of women for as long as 3 years.

  19. Intrinsic alterations in the partial molar volume on the protein denaturation: surficial Kirkwood-Buff approach.

    Science.gov (United States)

    Yu, Isseki; Takayanagi, Masayoshi; Nagaoka, Masataka

    2009-03-19

    The partial molar volume (PMV) of the protein chymotrypsin inhibitor 2 (CI2) was calculated by all-atom MD simulation. Denatured CI2 showed almost the same average PMV value as that of native CI2. This is consistent with the phenomenological question of the protein volume paradox. Furthermore, using the surficial Kirkwood-Buff approach, spatial distributions of PMV were analyzed as a function of the distance from the CI2 surface. The profiles of the new R-dependent PMV indicate that, in denatured CI2, the reduction in the solvent electrostatic interaction volume is canceled out mainly by an increment in thermal volume in the vicinity of its surface. In addition, the PMV of the denatured CI2 was found to increase in the region in which the number density of water atoms is minimum. These results provide a direct and detailed picture of the mechanism of the protein volume paradox suggested by Chalikian et al.

  20. Denaturing of plutonium by transmutation of minor-actinides for enhancement of proliferation resistance

    International Nuclear Information System (INIS)

    Sagara, Hiroshi; Saito, Masaki; Peryoga, Yoga; Ezoubtchenko, Alexey; Takivayev, Alan

    2005-01-01

    Feasibility study for the plutonium denaturing by utilizing minor-actinide transmutation in light water reactors has been performed. And the intrinsic feature of proliferation resistance of plutonium has been discussed based on IAEA's publication and Kessler's proposal. The analytical results show that not only 238 Pu but also other plutonium isotopes with even-mass-number have very important role for denaturing of plutonium due to their relatively large critical mass and noticeably high spontaneous fission neutron generation. With the change of the minor-actinide doping ratio in U-Pu mix oxide fuel and moderator to fuel ratio, it is found that the reactor-grade plutonium from conventional light water reactors can be denatured to satisfy the proliferation resistance criterion based on the Kessler's proposal but not to be sufficient for the criterion based on IAEA's publication. It has been also confirmed that all the safety coefficients take negative value throughout the irradiation. (author)

  1. 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.

  2. The fluid mechanics of continuous flow electrophoresis

    Science.gov (United States)

    Saville, D. A.

    1990-01-01

    The overall objective is to establish theoretically and confirm experimentally the ultimate capabilities of continuous flow electrophoresis chambers operating in an environment essentially free of particle sedimentation and buoyancy. The efforts are devoted to: (1) studying the effects of particle concentration on sample conductivity and dielectric constant. The dielectric constant and conductivity were identified as playing crucial roles in the behavior of the sample and on the resolving power and throughput of continuous flow devices; and (2) improving the extant mathematical models to predict flow fields and particle trajectories in continuous flow electrophoresis. A dielectric spectrometer was designed and built to measure the complex dielectric constant of a colloidal dispersion as a function of frequency between 500 Hz and 200 kHz. The real part of the signal can be related to the sample's conductivity and the imaginary part to its dielectric constant. Measurements of the dielectric constants of several different dispersions disclosed that the dielectric constants of dilute systems of the sort encountered in particle electrophoresis are much larger than would be expected based on the extant theory. Experiments were carried out to show that, in many cases, this behavior is due to the presence of a filamentary structure of small hairs on the particle surface. A technique for producing electrokinetically ideal synthetic latex particles by heat treating was developed. Given the ubiquitous nature of hairy surfaces with both cells and synthetic particles, it was deemed necessary to develop a theory to explain their behavior. A theory for electrophoretic mobility of hairy particles was developed. Finally, the extant computer programs for predicting the structure of electro-osmotically driven flows were extended to encompass flow channels with variable wall mobilities.

  3. Pulsed field gel electrophoresis a practical guide

    CERN Document Server

    Birren, Bruce

    1993-01-01

    Pulsed Field Gel Electrophoresis: A Practical Guide is the first laboratory manual to describe the theory and practice of this technique. Based on the authors' experience developing pulsed field gel instruments and teaching procedures, this book provides everything a researcher or student needs to know in order to understand and carry out pulsed field gel experiments. Clear, well-tested protocols assume only that users have a basic familiarity with molecular biology. Thorough coverage of useful data, theory, and applications ensures that this book is also a lasting resource for more adv

  4. Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

    Science.gov (United States)

    Bian, Liujiao; Ji, Xu

    2014-01-01

    Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

  5. The efficacy of denaturing actinide elements as a means of decreasing materials attractiveness

    Energy Technology Data Exchange (ETDEWEB)

    Hase, K.R.; Bathke, C.G. [Los Alamos National Laboratory: P.O. Box 1663, Los Alamos, NM 87545 (United States); Ebbinghaus, B.B.; Sleaford, B.W.; Robel, M. [Lawrence Livermore National Laboratory: P.O. Box 808, Livermore, CA 94551 (United States); Collins, B.A.; Prichard, A.W. [Pacific Northwest National Laboratory: P.O. Box 999, Richland, WA 99352 (United States)

    2013-07-01

    This study considers the concept of denaturing as applied to the actinide elements present in spent fuel as a means to reduce materials attractiveness. Highly attractive materials generally have low values of bare critical mass, heat content, and dose. To denature an attractive element, its spent-fuel isotopic composition (isotopic vector) is intentionally modified by introducing sufficient quantities of a significantly less attractive isotope to dilute the concentration of a highly attractive isotope so that the overall attractiveness of the element is reduced. The authors used FOM (Figure of Merit) formula as the material attractiveness metric for their parametric determination of the attractiveness of the Pu and U. Materials attractiveness needs to be considered in three distinct phases in the process to construct a nuclear explosive device (NED): the acquisition phase, processing phase, and utilization phase. The results show that denaturing uranium with {sup 238}U is actually an effective means of reducing the attractiveness. For uranium with a large minority of {sup 235}U, a mixture of 80% {sup 238}U to 20% {sup 235}U is required to reduce the attractiveness to low. For uranium with a large concentration of {sup 233}U, a mixture of 88% {sup 238}U to 12% {sup 233}U is required to reduce the attractiveness to low. The results also show that denaturing plutonium with {sup 238}Pu is less effective than denaturing uranium with {sup 238}U. Using {sup 238}Pu as the denaturing agent would require 80% or more by mass in order to reduce the attractiveness to low. No amount of {sup 240}Pu is enough to reduce the plutonium attractiveness below medium. The combination of {sup 238}Pu and {sup 240}Pu would require approximately 70% {sup 238}Pu and 25% {sup 240}Pu by mass to reduce the plutonium attractiveness to low.

  6. The efficacy of denaturing actinide elements as a means of decreasing materials attractiveness

    International Nuclear Information System (INIS)

    Hase, K.R.; Bathke, C.G.; Ebbinghaus, B.B.; Sleaford, B.W.; Robel, M.; Collins, B.A.; Prichard, A.W.

    2013-01-01

    This study considers the concept of denaturing as applied to the actinide elements present in spent fuel as a means to reduce materials attractiveness. Highly attractive materials generally have low values of bare critical mass, heat content, and dose. To denature an attractive element, its spent-fuel isotopic composition (isotopic vector) is intentionally modified by introducing sufficient quantities of a significantly less attractive isotope to dilute the concentration of a highly attractive isotope so that the overall attractiveness of the element is reduced. The authors used FOM (Figure of Merit) formula as the material attractiveness metric for their parametric determination of the attractiveness of the Pu and U. Materials attractiveness needs to be considered in three distinct phases in the process to construct a nuclear explosive device (NED): the acquisition phase, processing phase, and utilization phase. The results show that denaturing uranium with 238 U is actually an effective means of reducing the attractiveness. For uranium with a large minority of 235 U, a mixture of 80% 238 U to 20% 235 U is required to reduce the attractiveness to low. For uranium with a large concentration of 233 U, a mixture of 88% 238 U to 12% 233 U is required to reduce the attractiveness to low. The results also show that denaturing plutonium with 238 Pu is less effective than denaturing uranium with 238 U. Using 238 Pu as the denaturing agent would require 80% or more by mass in order to reduce the attractiveness to low. No amount of 240 Pu is enough to reduce the plutonium attractiveness below medium. The combination of 238 Pu and 240 Pu would require approximately 70% 238 Pu and 25% 240 Pu by mass to reduce the plutonium attractiveness to low

  7. Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing

    DEFF Research Database (Denmark)

    Masoud, Wafa Mahmoud Hasan; Takamiya, Monica K Wik; Vogensen, Finn Kvist

    2011-01-01

    ripening. Other bacteria like Corynebacterium, Halomonas, Pediococcus, Micrococcus and Staphylococcus, which were encountered in some cheese samples at low percentages compared with the total bacterial populations, were only detected by pyrosequencing. 16S rRNA gene pyrosequencing is an efficient method...

  8. Denaturing gradient gel electrophoresis-polymerase chain reaction comparison of chitosan effects on anaerobic cultures of broiler cecal bacteria and Salmonella Typhimurium

    Science.gov (United States)

    Salmonella colonization and product contamination are major poultry industry problems. Alternatives to traditional antibiotics against Salmonella offer the potential to lessen the development of resistance to antibiotics of importance to human health. The chitin derivative chitosan has drawn substa...

  9. Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments

    NARCIS (Netherlands)

    Garbeva, P.; Overbeek, van L.S.; Vuurde, van J.W.L.; Elsas, van J.D.

    2001-01-01

    The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desiree) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable

  10. Analysis of Fusarium populations in a soybean field under different fertilization management by real-time quantitative PCR and denaturing gradient gel electrophoresis

    Science.gov (United States)

    The abundance and population structure of Fusarium spp. in field soils were assessed to determine the effect of different fertilization treatments on soil microbial community and potential role in disease management. The field was under soybean-wheat-corn rotation located in the black soil (Udic Mo...

  11. Denaturing gradient gel electrophoresis analysis of fecal bacterial communities of marmot (Marmota marmota latirostris latirostris) in High Tatras (Slovakia) and their population fragmentation

    Czech Academy of Sciences Publication Activity Database

    Kopečný, Jan; Novotný, M.; Kmeť, V.

    2004-01-01

    Roč. 13, - (2004), s. 35-36 ISSN 1210-3209 Grant - others:Science and Technology Assistance Agency(SK) APVT-20-026102 Institutional research plan: CEZ:AV0Z5045916 Keywords : marmot * DGGE * Tatras Subject RIV: EE - Microbiology, Virology

  12. ANALYSIS OF BACTERIAL COMMUNITIES IN SEAGRASS BED SEDIMENTS BY DOUBLE-GRADIENT DENATURING GRADIENT GEL ELECTROPHORESIS OF PCR-AMPLIFIED 16SRRNA GENES

    Science.gov (United States)

    Bacterial communities associated with seagrass bed sediments are not well studied. The work presented here investigated several factors, including the presence or absence of vegetation, depth into sediment, and season, and their impact on bacterial community diversity. Double gra...

  13. Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

    Science.gov (United States)

    Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

    2016-02-01

    We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, pantibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

    Science.gov (United States)

    Forster, Ryan E.; Hert, Daniel G.; Chiesl, Thomas N.; Fredlake, Christopher P.; Barron, Annelise E.

    2009-01-01

    In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for “next-gen” sequencing platforms (e.g., the Illumina and 454 machines)—dsDNA molecules within a certain size range are “cut out” of a gel and recovered for subsequent “massively parallel” pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even “ultra-fast”), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro- or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications. PMID:19582705

  15. Micro-injector for capillary electrophoresis.

    Science.gov (United States)

    Sáiz, Jorge; Koenka, Israel Joel; García-Ruiz, Carmen; Müller, Beat; Chwalek, Thomas; Hauser, Peter C

    2015-08-01

    A novel micro-injector for capillary electrophoresis for the handling of samples with volumes down to as little as 300 nL was designed and built in our laboratory for analyses in which the available volume is a limitation. The sample is placed into a small cavity located directly in front of the separation capillary, and the injection is then carried out automatically by controlled pressurization of the chamber with compressed air. The system also allows automated flushing of the injection chamber as well as of the capillary. In a trial with a capillary electrophoresis system with contactless conductivity detector, employing a capillary of 25 μm diameter, the results showed good stability of migration times and peak areas. To illustrate the technique, the fast separation of five inorganic cations (Na(+) , K(+) , NH4 (+) , Ca(2+) , and Mg(2+) ) was set up. This could be achieved in less than 3 min, with good limits of detection (10 μM) and linear ranges (between about 10 and 1000 μM). The system was demonstrated for the determination of the inorganic cations in porewater samples of a lake sediment core. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Electrophoresis in the analysis of natural and industrial ob ects

    International Nuclear Information System (INIS)

    Stepanov, A.V.; Korchemnaya, E.K.

    1979-01-01

    Given is a brief review on practical application of electrophoresis in the analysis of natural and industrial objects. Suggested are expressiVe methods of thorium, uranium and rare earth elements separation in minerals by electrophoresis. The possibility of quantitative determination of rare earth elements in meteorites by the method of electromigration is shown. By means of electrophoresis identified are forms of radioruthenium in a sea water. Shown is the electrophoresis application for reactor loop water analysis, for environment contamination study, for determination of some rare earth yield in reactions of uranium fission by heavy ions

  17. 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.

  18. Reduction in beta-myosin heavy chains in stunned myocardium as assessed by nondenaturing gel electrophoresis.

    Science.gov (United States)

    Garcia, S C; Pomblum, V J; Gams, E; Rupp, H; Schipke, J D

    2007-09-01

    Myosin plays a key role in the structure and function of cardiac muscle. Three myosin isoenzymes (V(1), V(2), and V(3)) with different ATPase activities have been identified in mammalian ventricles based on their heavy chain constituents. The relative amount of myosin isoenzymes changes under physiological and pathological conditions. Until now, myosin isoenzymes have frequently been determined using either tube gel (nondenaturing) polyacrylamide gel electrophoresis (PAGE), or gradient or uniform sodium dodecyl sulfate (denaturing) PAGE. Both methods have disadvantages, e.g., a long running time. We developed, therefore, a uniform, nondenaturing PAGE with slab minigel format for analyzing the myosin isoenzymes in normoxic and stunned rabbit hearts. In normoxic hearts of adult rabbits, V(3) predominated over V(1) (46 vs 41%). In turn, in the stunned hearts, V(1) predominated over V(3) (70 vs 30%), and the heterodimeric V(2) was not anymore detectable. This alteration appears to result from a selective loss of myosin heavy chain (MHC)-beta. In parallel, the biochemical markers troponin I and creatine kinase were increased in the stunned hearts. We suggest that alterations of myosin isoenzymes in stunned myocardium can be monitored with native PAGE. The present analysis of myosin isoenzyme appears thus as a new tool for evaluating defects in MHC dimer formation in postischemic hearts.

  19. Colloid molecular weight estimation by gel chromatography/acrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Liberatore, F.A.; Dearborn, C.; Nigam, S.; Poon, C.; Camin, L.; Liteplo, M.

    1984-01-01

    Size or molecular weight (MW) estimation of radiolabeled collides in aqueous solutions has long been a problem. The authors have prepared several minimicroaggregated albumin colloids (mμAA) by heat denaturation of stannous-containing HSA solutions at pH 7.0, 7.5, and 8.5). The resulting colloids were labeled with Tc-99m and compared with Au-198 colloid and Tc-99m-antimony sulfide colloid (Tc-99m-Sb/sub 2/S3) by gel chromatography and gel electrophoresis. Tc-99mm-mμAA aggregated at pH 7.0 and the Au-198 colloid appeared in the external void volume of a BioRad A5.0 agarose column indicating an apparent MW of > 5 x 10/sup 6/ daltons. The pH7.5 Tc-99m-mμAA, migrated within the filtration range of the column as did a small fraction of Tc-99m-Sb/sub 2/S/sub 3/, suggesting that the MW is between 6 x 10/sup 4/ - 5 x 10/sup 6/ daltons. The Tc-99m-mμAA, aggregated at pH 8.5, had an apparent MW on gel filtration similar to that of untreated albumin, MW 6.6 x 10-/sup 4/ daltons. The mobilities of the colloids, on acrylamide disc gel electrophoresis, were consistent with the results on gel chromatography. The largest colloids, Au-198 colloid and pH 7.0 Tc-99m-mμAA, barely entered the separating gel; intermediate sized colloids, a small fraction of Tc-99m-Sb/sub 2/S/sub 3/ and pH 7.5 Tc-99m-mμAA migrated farther into the separating gel; while pH 8.5 Tc-99m-mμAA had mobility approaching that of untreated albumin. Lymphoscintigraphy studies using these colloids in animals showed the predicted, particle size-related differences in migration and clearance. The authors conclude that gel chromatography and gel electrophoresis are useful methods for estimating the apparent size of the colloidal particles

  20. Unravelling the hydrophobicity of urea in water using thermodiffusion: implications for protein denaturation.

    Science.gov (United States)

    Niether, Doreen; Di Lecce, Silvia; Bresme, Fernando; Wiegand, Simone

    2018-01-03

    Urea is widely used as a protein denaturant in aqueous solutions. Experimental and computer simulation studies have shown that it dissolves in water almost ideally at high concentrations, introducing little disruption in the water hydrogen bonded structure. However, at concentrations of the order of 5 M or higher, urea induces denaturation in a wide range of proteins. The origin of this behaviour is not completely understood, but it is believed to stem from a balance between urea-protein and urea-water interactions, with urea becoming possibly hydrophobic at a specific concentration range. The small changes observed in the water structure make it difficult to connect the denaturation effects to the solvation properties. Here we show that the exquisite sensitivity of thermodiffusion to solute-water interactions allows the identification of the onset of hydrophobicity of urea-water mixtures. The hydrophobic behaviour is reflected in a sign reversal of the temperature dependent slope of the Soret coefficient, which is observed, both in experiments and non-equilibrium computer simulations at ∼5 M concentration of urea in water. This concentration regime corresponds to the one where abrupt changes in the denaturation of proteins are commonly observed. We show that the onset of hydrophobicity is intrinsically connected to the urea-water interactions. Our results allow us to identify correlations between the Soret coefficient and the partition coefficient, log P, hence establishing the thermodiffusion technique as a powerful approach to study hydrophobicity.

  1. Thermal denaturation of protein studied by terahertz time-domain spectroscopy

    Science.gov (United States)

    Fu, Xiuhua; Li, Xiangjun; Liu, Jianjun; Du, Yong; Hong, Zhi

    2012-12-01

    In this study, the absorption spectra of native or thermal protein were measured in 0.2-1.4THz using terahertz time-domain spectroscopy (THz-TDS) system at room temperature, their absorption spectra and the refractive spectra were obtained. Experimental results indicate that protein both has strong absorption but their characteristics were not distinct in the THz region, and the absorption decreased during thermal denatured state. In order to prove protein had been denatured, we used Differential scanning calorimeter (DSC) measured their denatured temperature, from their DSC heating traces, collagen Td=101℃, Bovine serum albumin Td=97℃. While we also combined the Fourier transform infrared spectrometer (FTIR) to investigate their secondary and tertiary structure before and after denatuation, but the results did not have the distinct changes. We turned the absorption spectra and the refractive spectra to the dielectric spectra, and used the one-stage Debye model simulated the terahertz dielectric spectra of protein before and after denaturation. This research proved that the terahertz spectrum technology is feasible in testing protein that were affected by temperature or other factors which can provide theoretical foundation in the further study about the THz spectrum of protein and peptide temperature stability.

  2. Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison

    Energy Technology Data Exchange (ETDEWEB)

    Dahlen, Oda, E-mail: oda.dahlen@ntnu.no; Erp, Titus S. van, E-mail: titus.van.erp@ntnu.no [Department of Chemistry, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, Realfagbygget D3-117 7491 Trondheim (Norway)

    2015-06-21

    Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimental data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.

  3. Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

    International Nuclear Information System (INIS)

    Olive, P.L.; Vanderbyl, S.; MacPhail, S.H.

    1991-01-01

    Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though constraints to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to round up and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing

  4. 27 CFR 20.178 - Marks and brands on containers of specially denatured spirits.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Marks and brands on... Dealers § 20.178 Marks and brands on containers of specially denatured spirits. (a) Required marks. Each... officer, or (2) Consist of a brand name, or consist of caution notices, or consist of other material...

  5. Isoenergic modification of whey protein structure by denaturation and crosslinking using transglutaminase

    DEFF Research Database (Denmark)

    Stender, Emil G. P.; Koutina, Glykeria; Almdal, Kristoffer

    2018-01-01

    Transglutaminase (TG) catalyzes formation of covalent bonds between lysine and glutamine side chains and has applications in manipulation of food structure. Physical properties of a whey protein mixture (SPC) denatured either at elevated pH or by heat-treatment and followed by TG catalyzed...

  6. [Inactivating Effect of Heat-Denatured Lysozyme on Murine Norovirus in Bread Fillings].

    Science.gov (United States)

    Takahashi, Michiko; Yasuda, Yuka; Takahashi, Hajime; Takeuchi, Akira; Kuda, Takashi; Kimura, Bon

    2018-01-01

    In this study, we investigated the viability of murine norovirus strain 1 (MNV-1), a surrogate for human norovirus, in bread fillings used for making stuffed buns and pastries. The inactivating effect of heat-denatured lysozyme, which was recently reported to have an antiviral effect, on MNV-1 contaminating the bread fillings was also examined. MNV-1 was inoculated into two types of fillings (chocolate cream, marmalade jam) at 4.5 log PFU/g, and the bread fillings were stored at 4℃ for 5 days. MNV-1 remained viable in the bread fillings during storage. However, addition of 1% heat-denatured lysozyme to the fillings resulted in a decrease of MNV-1 infectivity immediately after inoculation, in both fillings. On the fifth day of storage, MNV-1 infectivity was decreased by 1.2 log PFU/g in chocolate cream and by 0.9 log PFU/g in marmalade jam. Although the mechanism underlying the anti-norovirus effect of heat-denatured lysozyme has not been clarified, our results suggest that heat-denatured lysozyme can be used as an inactivating agent against norovirus in bread fillings.

  7. Urea and Guanidinium Induced Denaturation of a Trp-Cage Miniprotein

    Czech Academy of Sciences Publication Activity Database

    Heyda, Jan; Kožíšek, Milan; Bednárová, Lucie; Thompson, G.; Konvalinka, Jan; Vondrášek, Jiří; Jungwirth, Pavel

    2011-01-01

    Roč. 115, č. 28 (2011), s. 8910-8924 ISSN 1520-6106 R&D Projects: GA MŠk LC512; GA ČR GA203/08/0114 Institutional research plan: CEZ:AV0Z40550506 Keywords : trp-cage denaturation * urea * guanidinium * molecular dynamics Subject RIV: CC - Organic Chemistry Impact factor: 3.696, year: 2011

  8. A Role For Ca 2+ in the Thermal and Urea Denaturation of ...

    African Journals Online (AJOL)

    Giant African snail (Achatina achatina) becomes dormant (aestivate) under harsh environmental conditions like dry seasons. During this period the animal accumulates urea and is faced with thermal death. The stability towards thermal and urea denaturation of haemocyanin from aestivating and nonaestivating A. achatina ...

  9. Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride.

    Science.gov (United States)

    Jha, Santosh Kumar; Marqusee, Susan

    2014-04-01

    Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).

  10. Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

    International Nuclear Information System (INIS)

    Ahluwalia, Unnati; Deep, Shashank

    2012-01-01

    Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

  11. Recent applications of nanomaterials in capillary electrophoresis.

    Science.gov (United States)

    González-Curbelo, Miguel Ángel; Varela-Martínez, Diana Angélica; Socas-Rodríguez, Bárbara; Hernández-Borges, Javier

    2017-10-01

    Nanomaterials have found an important place in Analytical Chemistry and, in particular, in Separation Science. Among them, metal-organic frameworks, magnetic and non-magnetic nanoparticles, carbon nanotubes and graphene, as well as their combinations, are the most important nanomaterials that have been used up to now. Concerning capillary electromigration techniques, these nanomaterials have also been used as both pseudostationary phases in electrokinetic chromatography (EKC) and as stationary phases in microchip capillary electrophoresis (CE) and capillary electrochromatography (CEC), as a result of their interesting and particular properties. This review article pretends to provide a general and critical revision of the most recent applications of nanomaterials in this field (period 2010-2017). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Electrophoresis in nanochannels: brief review and speculation

    Directory of Open Access Journals (Sweden)

    Santiago Juan G

    2006-11-01

    Full Text Available Abstract The relevant physical phenomena that dominate electrophoretic transport of ions and macromolecules within long, thin nanochannels are reviewed, and a few papers relevant to the discussion are cited. Sample ion transport through nanochannels is largely a function of their interaction with electric double layer. For small ions, this coupling includes the net effect of the external applied field, the internal field of the double layer, and the non-uniform velocity of the liquid. Adsorption/desorption kinetics and the effects of surface roughness may also be important in nanochannel electrophoresis. For macromolecules, the resulting motion is more complex as there is further coupling via steric interactions and perhaps polarization effects. These complex interactions and coupled physics represent a valuable opportunity for novel electrophoretic and chromatographic separations.

  13. Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

    Science.gov (United States)

    Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

    2015-01-01

    In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the

  14. Potential of capillary electrophoresis for the profiling of propolis

    NARCIS (Netherlands)

    Hilhorst, M.J; Somsen, G.W; de Jong, G.J.

    1998-01-01

    The usefulness of capillary electrophoresis (CE) with diode array detection for the profiling of Propolis, a hive product, is investigated. Water extracts of Propolis were analyzed with both capillary zone electrophoresis (CZE) at pH 7.0 and 9.3, and micellar electrokinetic chromatography (MEKC)

  15. Study of Streptavidin-Modified Quantum Dots by Capillary Electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Stanisavljevic, M.; Janů, L.; Šmerková, K.; Křížková, S.; Pizúrová, Naděžda; Ryvolová, M.; Adam, V.; Hubálek, J.; Kizek, R.

    2013-01-01

    Roč. 76, 7-8 (2013), s. 335-343 ISSN 0009-5893 Institutional support: RVO:68081723 Keywords : Capillary electrophoresis * Gel electrophoresis * Avidin-biotin technology * Oligonucleotide * Nanoparticle * quantum dots Subject RIV: CE - Biochemistry Impact factor: 1.370, year: 2013

  16. Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials

    Science.gov (United States)

    Ens, S.; Olson, A. B.; Dudley, C.; Ross, N. D., III; Siddiqi, A. A.; Umoh, K. M.; Schneegurt, M. A.

    2012-01-01

    Gel electrophoresis is the single most important molecular biology technique and it is central to life sciences research, but it is often too expensive for the secondary science classroom or homeschoolers. A simple safe low-cost procedure is described here that uses household materials to construct and run DNA gel electrophoresis. Plastic…

  17. 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…

  18. Polymer Nanocomposites

    Indian Academy of Sciences (India)

    methods for the synthesis of polymer nanocomposites. In this article we .... ers, raw materials recovery, drug delivery and anticorrosion .... region giving rise to dose-packed absorption bands called an IR ... using quaternary ammonium salts.

  19. Transurethral radiofrequency collagen denaturation for the treatment of women with urinary incontinence.

    Science.gov (United States)

    Kang, Diana; Han, Julia; Neuberger, Molly M; Moy, M Louis; Wallace, Sheila A; Alonso-Coello, Pablo; Dahm, Philipp

    2015-03-18

    Transurethral radiofrequency collagen denaturation is a relatively novel, minimally invasive device-based intervention used to treat individuals with urinary incontinence (UI). No systematic review of the evidence supporting its use has been published to date. To evaluate the efficacy of transurethral radiofrequency collagen denaturation, compared with other interventions, in the treatment of women with UI.Review authors sought to compare the following.• Transurethral radiofrequency collagen denaturation versus no treatment/sham treatment.• Transurethral radiofrequency collagen denaturation versus conservative physical treatment.• Transurethral radiofrequency collagen denaturation versus mechanical devices (pessaries for UI).• Transurethral radiofrequency collagen denaturation versus drug treatment.• Transurethral radiofrequency collagen denaturation versus injectable treatment for UI.• Transurethral radiofrequency collagen denaturation versus other surgery for UI. We conducted a systematic search of the Cochrane Incontinence Group Specialised Register (searched 19 December 2014), EMBASE and EMBASE Classic (January 1947 to 2014 Week 50), Google Scholar and three trials registries in December 2014, along with reference checking. We sought to identify unpublished studies by handsearching abstracts of major gynaecology and urology meetings, and by contacting experts in the field and the device manufacturer. Randomised and quasi-randomised trials of transurethral radiofrequency collagen denaturation versus no treatment/sham treatment, conservative physical treatment, mechanical devices, drug treatment, injectable treatment for UI or other surgery for UI in women were eligible. We screened search results and selected eligible studies for inclusion. We assessed risk of bias and analysed dichotomous variables as risk ratios (RRs) with 95% confidence intervals (CIs) and continuous variables as mean differences (MDs) with 95% CIs. We rated the quality of

  20. DNA gel electrophoresis: the reptation model(s).

    Science.gov (United States)

    Slater, Gary W

    2009-06-01

    DNA gel electrophoresis has been the most important experimental tool to separate DNA fragments for several decades. The introduction of PFGE in the 1980s and capillary gel electrophoresis in the 1990s made it possible to study, map and sequence entire genomes. Explaining how very large DNA molecules move in a gel and why PFGE is needed to separate them has been an active field of research ever since the launch of the journal Electrophoresis. This article presents a personal and historical overview of the development of the theory of gel electrophoresis, focusing on the reptation model, the band broadening mechanisms, and finally the factors that limit the read length and the resolution of electrophoresis-based sequencing systems. I conclude with a short discussion of some of the questions that remain unanswered.

  1. Agarose gel electrophoresis and polyacrylamide gel electrophoresis for visualization of simple sequence repeats.

    Science.gov (United States)

    Anderson, James; Wright, Drew; Meksem, Khalid

    2013-01-01

    In the modern age of genetic research there is a constant search for ways to improve the efficiency of plant selection. The most recent technology that can result in a highly efficient means of selection and still be done at a low cost is through plant selection directed by simple sequence repeats (SSRs or microsatellites). The molecular markers are used to select for certain desirable plant traits without relying on ambiguous phenotypic data. The best way to detect these is the use of gel electrophoresis. Gel electrophoresis is a common technique in laboratory settings which is used to separate deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by size. Loading DNA and RNA onto gels allows for visualization of the size of fragments through the separation of DNA and RNA fragments. This is achieved through the use of the charge in the particles. As the fragments separate, they form into distinct bands at set sizes. We describe the ability to visualize SSRs on slab gels of agarose and polyacrylamide gel electrophoresis.

  2. Quantitative analysis by microchip capillary electrophoresis – current limitations and problem-solving strategies

    NARCIS (Netherlands)

    Revermann, T.; Götz, S.; Künnemeyer, Jens; Karst, U.

    2008-01-01

    Obstacles and possible solutions for the application of microchip capillary electrophoresis in quantitative analysis are described and critically discussed. Differences between the phenomena occurring during conventional capillary electrophoresis and microchip-based capillary electrophoresis are

  3. Polymer nanocomposites: polymer and particle dynamics

    KAUST Repository

    Kim, Daniel; Srivastava, Samanvaya; Narayanan, Suresh; Archer, Lynden A.

    2012-01-01

    Polymer nanocomposites containing nanoparticles smaller than the random coil size of their host polymer chains are known to exhibit unique properties, such as lower viscosity and glass transition temperature relative to the neat polymer melt. It has

  4. Antimocrobial Polymer

    Science.gov (United States)

    McDonald, William F.; Huang, Zhi-Heng; Wright, Stacy C.

    2005-09-06

    A polymeric composition having antimicrobial properties and a process for rendering the surface of a substrate antimicrobial are disclosed. The composition comprises a crosslinked chemical combination of (i) a polymer having amino group-containing side chains along a backbone forming the polymer, (ii) an antimicrobial agent selected from quaternary ammonium compounds, gentian violet compounds, substituted or unsubstituted phenols, biguanide compounds, iodine compounds, and mixtures thereof, and (iii) a crosslinking agent containing functional groups capable of reacting with the amino groups. In one embodiment, the polymer is a polyamide formed from a maleic anhydride or maleic acid ester monomer and alkylamines thereby producing a polyamide having amino substituted alkyl chains on one side of the polyamide backbone; the crosslinking agent is a phosphine having the general formula (A)3P wherein A is hydroxyalkyl; and the antimicrobial agent is chlorhexidine, dimethylchlorophenol, cetyl pyridinium chloride, gentian violet, triclosan, thymol, iodine, and mixtures thereof.

  5. Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

    Science.gov (United States)

    Kim, Jeongyong; Song, Hugeun; Park, Inho; Carlisle, Christine R; Bonin, Keith; Guthold, Martin

    2011-03-01

    Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers. Copyright © 2010 Wiley-Liss, Inc.

  6. Theoretical aspects of pressure and solute denaturation of proteins: A Kirkwood-buff-theory approach

    Science.gov (United States)

    Ben-Naim, Arieh

    2012-12-01

    A new approach to the problem of pressure-denaturation (PD) and solute-denaturation (SD) of proteins is presented. The problem is formulated in terms of Le Chatelier principle, and a solution is sought in terms of the Kirkwood-Buff theory of solutions. It is found that both problems have one factor in common; the excluded volumes of the folded and the unfolded forms with respect to the solvent molecules. It is shown that solvent-induced effects operating on hydrophilic groups along the protein are probably the main reason for PD. On the other hand, the SD depends on the preferential solvation of the folded and the unfolded forms with respect to solvent and co-solvent molecules.

  7. Teaching what one does not know: strangeness and denaturation in (autobiographical narrations

    Directory of Open Access Journals (Sweden)

    Jorge Luiz da Cunha

    2014-01-01

    Full Text Available The thematic focus in this text are the estrangement/denaturation processes in (autobiographical narrations. The aim of this study was to reflect on the possibility to promote estrangement/denatura - tion in (autobiographical writings made by teenagers in the space/ time of the classroom environment. The methodological proposal consisted on developing (autobiographical writings by students from sociology classes in High School. A total of 138 teenagers from a public school, attending the first school trimester in the year 2013, have participated in the study. The concepts of estrangement/de - naturation are located in the anthropology field and, the work with (autobiographical narrations is located in the socio-clinic perspec - tives and of biographization processes. The results indicate that (autobiographical narrations provide estrangements/denaturation and go towards teaching what one does not know. We can, then, conclude that this possibility, as an educational act, may generate knowledge suspension to self-inventiveness.

  8. Two-dimensional salt and temperature DNA denaturation analysis using a magnetoresistive sensor

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Dufva, Martin; Hansen, Mikkel Fougt

    2017-01-01

    We present a microfluidic system and its use to measure DNA denaturation curves by varying the temperature or salt (Na+) concentration. The readout is based on real-time measurements of DNA hybridization using magnetoresistive sensors and magnetic nanoparticles (MNPs) as labels. We report the first...... melting curves of DNA hybrids measured as a function of continuously decreasing salt concentration at fixed temperature and compare them to the corresponding curves obtained vs. temperature at fixed salt concentration. The magnetoresistive sensor platform provided reliable results under varying....... The results demonstrate that concentration melting provides an attractive alternative to temperature melting in on-chip DNA denaturation experiments and further show that the magnetoresistive platform is attractive due to its low cross-sensitivity to temperature and liquid composition....

  9. Ceramic protective coatings applied by sol-gel or electrophoresis

    International Nuclear Information System (INIS)

    Stoch, A.

    1993-01-01

    Sol-gel and electrophoresis are the complementary techniques which may be used for obtaining the ceramic coatings. The composition of such a coatings depends on the composition of electrophoresis bath or sol solution. Thermal treatment is used for densifying the coating and promoting the adherence of coating to the substrate. In presented work silica, silica-alumina or alumina coatings are applied by sol-gel dip coating procedure on steel, aluminium or ceramic substrates. Electrophoresis is employed for obtaining zirconia, alumina or hydroxyapatite coatings on stainless steel. (author). 7 refs

  10. Basic study for gas cleaning using discharge and electrophoresis

    International Nuclear Information System (INIS)

    Su, Zhen-Zhou; Sawada, Jun; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2004-01-01

    A NO x removal method using discharge plasma and electrophoresis for exhaust control was studied. The 65-50% of NO was oxidized to NO 2 or HNO 3 by the discharge plasma with specific input energy of 45J/l. The electrophoresis was carried out to concentrate the NO 2 or HNO 3 adsorbed on the adsorbents. As a result, 80% of the adsorbed nitrate ions were found in the anode region. A combination of molecular sieve pellets of 13X and glass fiber cloth was tested for the collection of nitrate ions. The ability of simultaneous concentration of nitrate ions and sulfate ions using electrophoresis was examined

  11. Simulating Electrophoresis with Discrete Charge and Drag

    Science.gov (United States)

    Mowitz, Aaron J.; Witten, Thomas A.

    A charged asymmetric rigid cluster of colloidal particles in saline solution can respond in exotic ways to an electric field: it may spin or move transversely. These distinctive motions arise from the drag force of the neutralizing countercharge surrounding the cluster. Because of this drag, calculating the motion of arbitrary asymmetric objects with nonuniform charge is impractical by conventional methods. Here we present a new method of simulating electrophoresis, in which we replace the continuous object and the surrounding countercharge with discrete point-draggers, called Stokeslets. The balance of forces imposes a linear, self-consistent relation among the drag and Coulomb forces on the Stokeslets, which allows us to easily determine the object's motion via matrix inversion. By explicitly enforcing charge+countercharge neutrality, the simulation recovers the distinctive features of electrophoretic motion to few-percent accuracy using as few as 1000 Stokeslets. In particular, for uniformly charged objects, we observe the characteristic Smoluchowski independence of mobility on object size and shape. We then discuss electrophoretic motion of asymmetric objects, where our simulation method is particularly advantageous. This work is supported by a Grant from the US-Israel Binational Science Foundation.

  12. Active airborne contamination control using electrophoresis

    International Nuclear Information System (INIS)

    Veatch, B.D.

    1994-01-01

    In spite of our best efforts, radioactive airborne contamination continues to be a formidable problem at many of the Department of Energy (DOE) weapons complex sites. For workers that must enter areas with high levels of airborne contamination, personnel protective equipment (PPE) can become highly restrictive, greatly diminishing productivity. Rather than require even more restrictive PPE for personnel in some situations, the Rocky Flats Plant (RFP) is actively researching and developing methods to aggressively combat airborne contamination hazards using electrophoretic technology. With appropriate equipment, airborne particulates can be effectively removed and collected for disposal in one simple process. The equipment needed to implement electrophoresis is relatively inexpensive, highly reliable, and very compact. Once airborne contamination levels are reduced, less PPE is required and a significant cost savings may be realized through decreased waste and maximized productivity. Preliminary ''cold,'' or non-radioactive, testing results at the RFP have shown the technology to be effective on a reasonable scale, with several potential benefits and an abundance of applications

  13. Van de Graaff generator for capillary electrophoresis.

    Science.gov (United States)

    Lee, Seung Jae; Castro, Eric R; Guijt, Rosanne M; Tarn, Mark D; Manz, Andreas

    2017-09-29

    A new approach for high voltage capillary electrophoresis (CE) is proposed, which replaces the standard high voltage power supply with a Van de Graaff generator, a low current power source. Because the Van de Graaff generator is a current-limited source (10μA), potentials exceeding 100kV can be generated for CE when the electrical resistance of the capillary is maximized. This was achieved by decreasing the capillary diameter and reducing the buffer ionic strength. Using 2mM borate buffer and a 5μm i.d. capillary, fluorescently labeled amino acids were separated with efficiencies up to 3.5 million plates; a 5.7 fold improvement in separation efficiency compared to a normal power supply (NPS) typically used in CE. This separation efficiency was realized using a simple set-up without significant Joule heating, making the Van de Graaff generator a promising alternative for applying the high potentials required for enhancing resolution in the separation and analysis of highly complex samples, for example mixtures of glycans. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Fabricating PFPE Membranes for Capillary Electrophoresis

    Science.gov (United States)

    Lee, Michael C.; Willis, Peter A.; Greer, Frank; Rolland, Jason

    2009-01-01

    A process has been developed for fabricating perfluoropolyether (PFPE) membranes that contain microscopic holes of precise sizes at precise locations. The membranes are to be incorporated into laboratory-on-a-chip microfluidic devices to be used in performing capillary electrophoresis. The present process is a modified version of part of the process, described in the immediately preceding article, that includes a step in which a liquid PFPE layer is cured into solid (membrane) form by use of ultraviolet light. In the present process, one exploits the fact that by masking some locations to prevent exposure to ultraviolet light, one can prevent curing of the PFPE in those locations. The uncured PFPE can be washed away from those locations in the subsequent release and cleaning steps. Thus, holes are formed in the membrane in those locations. The most straightforward way to implement the modification is to use, during the ultraviolet-curing step, an ultraviolet photomask similar to the photomasks used in fabricating microelectronic devices. In lieu of such a photomask, one could use a mask made of any patternable ultraviolet-absorbing material (for example, an ink or a photoresist).

  15. Native and denatured bovine serum albumin. D.c. polarography, stripping voltammetry and constant current chronopotentiometry

    Czech Academy of Sciences Publication Activity Database

    Ostatná, Veronika; Uslu, B.; Dogan, B.; Ozkan, S.; Paleček, Emil

    2006-01-01

    Roč. 593, č. 1-2 (2006), s. 172-178 ISSN 0022-0728 R&D Projects: GA AV ČR(CZ) IAA500040513; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507 Keywords : protein electrochemistry * bovine serum albumin * native and denatured proteins Subject RIV: BO - Biophysics Impact factor: 2.339, year: 2006

  16. Micro-CT Imaging of Denatured Chitin by Silver to Explore Honey Bee and Insect Pathologies

    Science.gov (United States)

    Butzloff, Peter R.

    2011-01-01

    Background Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term “denatured chitin” calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. Methodology/Principal Findings A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. Conclusions/Significance The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an

  17. Micro-CT imaging of denatured chitin by silver to explore honey bee and insect pathologies.

    Directory of Open Access Journals (Sweden)

    Peter R Butzloff

    Full Text Available BACKGROUND: Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term "denatured chitin" calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. METHODOLOGY/PRINCIPAL FINDINGS: A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT. Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi, at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. CONCLUSIONS/SIGNIFICANCE: The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may

  18. Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes

    Czech Academy of Sciences Publication Activity Database

    Ostatná, Veronika; Černocká, Hana; Kurzatkowska, K.; Paleček, Emil

    2012-01-01

    Roč. 735, JUL (2012), s. 31-36 ISSN 0003-2670 R&D Projects: GA AV ČR(CZ) KJB100040901; GA ČR(CZ) GAP301/11/2055; GA MŠk(CZ) ME09038 Institutional research plan: CEZ:AV0Z50040702 Keywords : protein denaturation * carbon electrodes * edge plane pyrolytic graphite Subject RIV: BO - Biophysics Impact factor: 4.387, year: 2012

  19. Multifocal peritoneal splenosis in Tc-99m-labeled heat-denatured red blood cell scintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Min Ki; Hwang, Kyung Hoon; Choe, Won Sick [Gachon University Gil Medical Center, Incheon (Korea, Republic of)

    2006-06-15

    A 44-year-old man with a past medical history of splenectomy came to hospital because of epigastric pain abdominopelvic computed tomography(CT) showed a soft tissue mass and multifocal variable-sized nodules as well as finding suggestive of cholecystitis. Subsequently, he underwent Tc-99m-labeled heat- denatured red blood cell(RBC) scintigraphy to evaluate the mass and nodules. The scintigraphy confirmed multifocal peritoneal splenosis in the abdominopelvic cavity.

  20. Equilibrium unfolding of A. niger RNase: pH dependence of chemical and thermal denaturation.

    Science.gov (United States)

    Kumar, Gundampati Ravi; Sharma, Anurag; Kumari, Moni; Jagannadham, Medicherla V; Debnath, Mira

    2011-08-01

    Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A(MG1)) into another (I(MG2)).

  1. Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes

    2004-02-09

    Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light ({lambda}{sub max}=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -1}. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in {beta}-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.

  2. Polymer electronics

    CERN Document Server

    Geoghegan, Mark

    2013-01-01

    Polymer electronics is the science behind many important new developments in technology, such as the flexible electronic display (e-ink) and many new developments in transistor technology. Solar cells, light-emitting diodes, and transistors are all areas where plastic electronics is likely to, or is already having, a serious impact on our daily lives. With polymer transistors and light-emitting diodes now being commercialised, there is a clear need for a pedagogic text thatdiscusses the subject in a clear and concise fashion suitable for senior undergraduate and graduate students. The content

  3. Denaturation of proteins by surfactants studied by the Taylor dispersion analysis.

    Directory of Open Access Journals (Sweden)

    Aldona Jelińska

    Full Text Available We showed that the Taylor Dispersion Analysis (TDA is a fast and easy to use method for the study of denaturation proteins. We applied TDA to study denaturation of β-lactoglobulin, transferrin, and human insulin by anionic surfactant sodium dodecyl sulfate (SDS. A series of measurements at constant protein concentration (for transferrin was 1.9 x 10-5 M, for β- lactoglobulin was 7.6 x 10-5 M, and for insulin was 1.2 x 10-4 M and varying SDS concentrations were carried out in the phosphate-buffered saline (PBS. The structural changes were analyzed based on the diffusion coefficients of the complexes formed at various surfactant concentrations. The concentration of surfactant was varied in the range from 1.2 x 10-4 M to 8.7 x 10-2 M. We determined the minimum concentration of the surfactant necessary to change the native conformation of the proteins. The minimal concentration of SDS for β-lactoglobulin and transferrin was 4.3 x 10-4 M and for insulin 2.3 x 10-4 M. To evaluate the TDA as a novel method for studying denaturation of proteins we also applied other methods i.e. electronic circular dichroism (ECD and dynamic light scattering (DLS to study the same phenomenon. The results obtained using these methods were in agreement with the results from TDA.

  4. Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

    Science.gov (United States)

    Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

    2017-08-08

    To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

  5. Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

    Science.gov (United States)

    Siepi, Marialuisa; Politi, Jane; Dardano, Principia; Amoresano, Angela; De Stefano, Luca; Monti, Daria Maria; Notomista, Eugenio

    2017-08-01

    Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes.

  6. Evaluation of gasoline-denatured ethanol as a carbon source for denitrification.

    Science.gov (United States)

    Kazasi, Anna; Boardman, Gregory D; Bott, Charles B

    2013-06-01

    In this study concerning denitrification, the performance of three carbon sources, methanol (MeOH), ethanol (EtOH) and gasoline-denatured ethanol (dEtOH), was compared and evaluated on the basis of treatment efficiency, inhibition potential and cost. The gasoline denaturant considered here contained mostly aliphatic compounds and little of the components that typically boost the octane rating, such as benzene, toluene, ethylbenzene and xylenes. Results were obtained using three lab-scale SBRs operated at SRT of 12.0 +/- 0.9 days. After biomass was acclimated, denitrification rates with dEtOH were similar to those of EtOH (201 +/- 50 and 197 +/- 28 NO3-N/g MLVSS x d, respectively), and higher than those of MeOH (165 +/- 49 mg NO3-N/g MLVSS x d). The denaturant did not affect biomass production, nitrification or denitrification. Effluent soluble COD concentrations were always less than the analytical detection limit. Although the cost of dEtOH ($2.00/kg nitrate removed) was somewhat higher than that of methanol ($1.63/kg nitrate removed), the use of dEtOH is very promising and utilities will have to decide if it is worth paying a little extra to take advantage of its benefits.

  7. Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

    Science.gov (United States)

    Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

    2013-05-01

    In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Copyright © 2013 Wiley Periodicals, Inc.

  8. Radioimmunoassay and heat denaturation enzyme assay for the detection of Tay-Sachs heterozygotes during pregnancy

    International Nuclear Information System (INIS)

    Nguyen, C.; Gold, R.J.M.; Mahuran, D.; Lowden, J.A.

    1981-01-01

    Tay-Sachs disease results from a loss of activity of hexosaminidase A (HEX A) in body tissues and fluids. Heterozygotes for the disease are usually identified by their relatively low ratio of heat-labile HEX A to total hexosaminidase. During pregnancy an intermediate isoenzyme (HEX I) increases in activity in serum and obscures the heterozygote status. HEX I does not increase in leucocytes, tears and other body tissues but because of technical difficulties in these assays the authors examined the feasibility of using a radioimmunoassay for HEX A. By univariate analysis, the heat denaturation assay gave a lower cost of misclassification for non-pregnant normals while RIA did so for pregnant normals. A combination of both tests led to reduced cost of misclassification compared to either alone. Bayesian analysis of bivariate gaussian density functions for heat denaturation and for radioimmunoassays of HEX isoenzymes was employed to calculate misclassification frequencies. Among the parameters examined, HEX A measured by RIA and % HEX A by heat-denaturation assay were the two having the best discriminatory power. (Auth.)

  9. Isothermal chemical denaturation of large proteins: Path-dependence and irreversibility.

    Science.gov (United States)

    Wafer, Lucas; Kloczewiak, Marek; Polleck, Sharon M; Luo, Yin

    2017-12-15

    State functions (e.g., ΔG) are path independent and quantitatively describe the equilibrium states of a thermodynamic system. Isothermal chemical denaturation (ICD) is often used to extrapolate state function parameters for protein unfolding in native buffer conditions. The approach is prudent when the unfolding/refolding processes are path independent and reversible, but may lead to erroneous results if the processes are not reversible. The reversibility was demonstrated in several early studies for smaller proteins, but was assumed in some reports for large proteins with complex structures. In this work, the unfolding/refolding of several proteins were systematically studied using an automated ICD instrument. It is shown that: (i) the apparent unfolding mechanism and conformational stability of large proteins can be denaturant-dependent, (ii) equilibration times for large proteins are non-trivial and may introduce significant error into calculations of ΔG, (iii) fluorescence emission spectroscopy may not correspond to other methods, such as circular dichroism, when used to measure protein unfolding, and (iv) irreversible unfolding and hysteresis can occur in the absence of aggregation. These results suggest that thorough confirmation of the state functions by, for example, performing refolding experiments or using additional denaturants, is needed when quantitatively studying the thermodynamics of protein unfolding using ICD. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Effects of high pressure freezing (HPF) on denaturation of natural actomyosin extracted from prawn (Metapenaeus ensis).

    Science.gov (United States)

    Cheng, Lina; Sun, Da-Wen; Zhu, Zhiwei; Zhang, Zhihang

    2017-08-15

    Effects of protein denaturation caused by high pressure freezing, involving Pressure-Factors (pressure, time) and Freezing-Factors (temperature, phase transition, recrystallization, ice crystal types), are complicated. In the current study, the conformation and functional changes of natural actomyosin (NAM) under pressure assisted freezing (PAF, 100,150,300,400,500MPa P -20°C/25min ), pressure shift freezing (PSF, 200MPa P -20°C/25min ), and immersion freezing ( 0.1MPa P -20°C/5min ) after pressure was released to 0.1MPa, as compared to normal immersion freezing process (IF, 0.1MPa P -20°C/30min ). Results indicated that PSF ( 200MPa P -20°C/30min ) could reduce the denaturation of frozen NAM and a pressure of 300MPa was the critical point to induce such a denaturation. During the periods of B→D in PSF or B→C→D in PAF, the generation and growth of ice crystals played an important role on changing the secondary and tertiary structure of the treated NAM. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis.

    Science.gov (United States)

    Mozafari, Mona; El Deeb, Sami; Krull, Friederike; Wildgruber, Robert; Weber, Gerhard; Reiter, Christian G; Wätzig, Hermann

    2018-02-01

    A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis.

    Science.gov (United States)

    Kooy, Floor K; Ma, Muyuan; Beeftink, Hendrik H; Eggink, Gerrit; Tramper, Johannes; Boeriu, Carmen G

    2009-01-15

    Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

  13. Determination of size distribution of small DNA fragments by polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Lau How Mooi

    1998-01-01

    Size distribution determination of DNA fragments can be normally determined by the agarose gel electrophoresis, including the normal DNA banding pattern analysis. However this method is only good for large DNA, such as the DNA of the size of kilo base pairs to mega base pairs range. DNA of size less than kilo base pairs is difficult to be quantified by the agarose gel method. Polyacrylamide gel electrophoresis however can be used to measure the quantity of DNA fragments of size less than kilo base pairs in length, down to less than ten base pairs. This method is good for determining the quantity of the smaller size DNA, single stranded polymers or even some proteins, if the known standards are available. In this report detail description of the method of preparing the polyacrylamide gel, and the experimental set up is discussed. Possible uses of this method, and the comparison with the standard sizes of DNA is also shown. This method is used to determine the distribution of the amount of the fragmented DNA after the Calf-thymus DNA has been exposed to various types of radiation and of different doses. The standards were used to determine the sizes of the fragmented Calf-thymus DNA. The higher the dose the higher is the amount of the smaller size DNA measured

  14. Polymer physics

    CERN Document Server

    Gedde, Ulf W

    1999-01-01

    This book is the result of my teaching efforts during the last ten years at the Royal Institute of Technology. The purpose is to present the subject of polymer physics for undergraduate and graduate students, to focus the fundamental aspects of the subject and to show the link between experiments and theory. The intention is not to present a compilation of the currently available literature on the subject. Very few reference citations have thus been made. Each chapter has essentially the same structure: starling with an introduction, continuing with the actual subject, summarizing the chapter in 30D-500 words, and finally presenting problems and a list of relevant references for the reader. The solutions to the problems presented in Chapters 1-12 are given in Chapter 13. The theme of the book is essentially polymer science, with the exclusion of that part dealing directly with chemical reactions. The fundamentals in polymer science, including some basic polymer chemistry, are presented as an introduction in t...

  15. Antimicrobial polymers.

    Science.gov (United States)

    Jain, Anjali; Duvvuri, L Sailaja; Farah, Shady; Beyth, Nurit; Domb, Abraham J; Khan, Wahid

    2014-12-01

    Better health is basic requirement of human being, but the rapid growth of harmful pathogens and their serious health effects pose a significant challenge to modern science. Infections by pathogenic microorganisms are of great concern in many fields such as medical devices, drugs, hospital surfaces/furniture, dental restoration, surgery equipment, health care products, and hygienic applications (e.g., water purification systems, textiles, food packaging and storage, major or domestic appliances etc.) Antimicrobial polymers are the materials having the capability to kill/inhibit the growth of microbes on their surface or surrounding environment. Recently, they gained considerable interest for both academic research and industry and were found to be better than their small molecular counterparts in terms of enhanced efficacy, reduced toxicity, minimized environmental problems, resistance, and prolonged lifetime. Hence, efforts have focused on the development of antimicrobial polymers with all desired characters for optimum activity. In this Review, an overview of different antimicrobial polymers, their mechanism of action, factors affecting antimicrobial activity, and application in various fields are given. Recent advances and the current clinical status of these polymers are also discussed. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  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. TECHNIQUES WITH POTENTIAL FOR HANDLING ENVIRONMENTAL SAMPLES IN CAPILLARY ELECTROPHORESIS

    Science.gov (United States)

    An assessment of the methods for handling environmental samples prior to capillary electrophoresis (CE) is presented for both aqueous and solid matrices. Sample handling in environmental analyses is the subject of ongoing research at the Environmental Protection Agency's National...

  18. A new electrophoresis technique to separate microsatellite alleles ...

    African Journals Online (AJOL)

    A new electrophoresis technique to separate microsatellite alleles* ... African Journal of Biotechnology ... with the CEQTM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer easily separated products and determined allelic size, ...

  19. Capillary electrophoresis in the N-glycosylation analysis of biopharmaceuticals

    Czech Academy of Sciences Publication Activity Database

    Guttman, András

    2013-01-01

    Roč. 48, JUL-AUG (2013), s. 132-143 ISSN 0165-9936 Institutional support: RVO:68081715 Keywords : automated workflow * biopharmaceuticals * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 6.612, year: 2013

  20. Success and failure with phthalate buffers in capillary zone electrophoresis

    NARCIS (Netherlands)

    Bocek, P.; Gebauer, P.; Beckers, J.L.

    2001-01-01

    Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the

  1. Characterization of biological macromolecules by electrophoresis and neutron activation

    International Nuclear Information System (INIS)

    Stone, S.F.; Hancock, D.; Zeisler, R.

    1987-01-01

    A procedure combining polyacrylamide gel electrophoresis (PAGE) with INAA and autoradiography was developed to study biological macromolecules and their associated trace elements. Results from the application of this method to several metalloproteins are presented. (author)

  2. Methods and instrumentation for quantitative microchip capillary electrophoresis

    NARCIS (Netherlands)

    Revermann, T.

    2007-01-01

    The development of novel instrumentation and analytical methodology for quantitative microchip capillary electrophoresis (MCE) is described in this thesis. Demanding only small quantities of reagents and samples, microfluidic instrumentation is highly advantageous. Fast separations at high voltages

  3. Exact method for numerically analyzing a model of local denaturation in superhelically stressed DNA

    International Nuclear Information System (INIS)

    Fye, R.M.; Benham, C.J.

    1999-01-01

    Local denaturation, the separation at specific sites of the two strands comprising the DNA double helix, is one of the most fundamental processes in biology, required to allow the base sequence to be read both in DNA transcription and in replication. In living organisms this process can be mediated by enzymes which regulate the amount of superhelical stress imposed on the DNA. We present a numerically exact technique for analyzing a model of denaturation in superhelically stressed DNA. This approach is capable of predicting the locations and extents of transition in circular superhelical DNA molecules of kilobase lengths and specified base pair sequences. It can also be used for closed loops of DNA which are typically found in vivo to be kilobases long. The analytic method consists of an integration over the DNA twist degrees of freedom followed by the introduction of auxiliary variables to decouple the remaining degrees of freedom, which allows the use of the transfer matrix method. The algorithm implementing our technique requires O(N 2 ) operations and O(N) memory to analyze a DNA domain containing N base pairs. However, to analyze kilobase length DNA molecules it must be implemented in high precision floating point arithmetic. An accelerated algorithm is constructed by imposing an upper bound M on the number of base pairs that can simultaneously denature in a state. This accelerated algorithm requires O(MN) operations, and has an analytically bounded error. Sample calculations show that it achieves high accuracy (greater than 15 decimal digits) with relatively small values of M (M<0.05N) for kilobase length molecules under physiologically relevant conditions. Calculations are performed on the superhelical pBR322 DNA sequence to test the accuracy of the method. With no free parameters in the model, the locations and extents of local denaturation predicted by this analysis are in quantitatively precise agreement with in vitro experimental measurements

  4. Phylogenetic reconstruction of South American felids defined by protein electrophoresis

    OpenAIRE

    Pecon Slattery, J.; Johnson, W. E.; Goldman, D.; O'Brien, S. J.

    1994-01-01

    Phylogenetic associations among six closely related South American felid species were defined by changes in protein-encoding gene loci. We analyzed proteins isolated from skin fibroblasts using two-dimensional electrophoresis and allozymes extracted from blood cells. Genotypes were determined for multiple individuals of ocelot, margay, tigrina, Geoffroy's cat, kodkod, and pampas cat at 548 loci resolved by two-dimensional electrophoresis and 44 allozyme loci. Phenograms were constructed using...

  5. Field-portable Capillary Electrophoresis Instrument with Conductivity Detection

    International Nuclear Information System (INIS)

    Zhang, H F; Liu, X W; Wang, W; Wang, X L; Tian, L

    2006-01-01

    In this paper a novel capillary electrophoresis chip (CEC) is presented with integrated platinum electrodes and simplified conductivity detector. CEC is fabricated by the method of mechanical modification with probe on organic glass. Capillary electrophoresis chip can rapidly completed ion separation by simulation of concentration distribution and zone-broadening. Detection circuit is simple which can detect pA order current. This system has those advantages such as small volume, low power consumption and linearity, and well suit for field analysis

  6. Capillary Electrophoresis Artifact Due to Eosin

    Science.gov (United States)

    Murphy, Kathleen M.; Berg, Karin D.; Geiger, Tanya; Hafez, Michael; Flickinger, Katie A.; Cooper, Lisa; Pearson, Patrick; Eshleman, James R.

    2005-01-01

    Capillary electrophoresis (CE) is a commonly used tool in the analysis of fluorescently labeled PCR amplification products. We have identified a CE artifact caused by the tissue stain eosin that can complicate the interpretation of CE data. The artifact was detected during routine analysis of a DNA sample isolated from a formalin-fixed, paraffin-embedded tissue sample considered histologically suspicious for a B-cell neoplasm. A standard clinical PCR and CE assay for immunoglobulin heavy chain (IGH) gene rearrangement revealed a weak polyclonal population of rearranged IGH genes and a 71 base peak suspicious for IGH clonality. The spectral properties of the 71 base peak were unusual in that although the dominant fluorescence of the peak was blue, it also fluoresced in green and yellow (blue>green>yellow), raising the suspicion that the peak might represent an artifact. CE analysis of the genomic DNA sample without PCR amplification demonstrated the presence of the 71 base peak, suggesting that the artifact was caused by a contaminant within the DNA sample itself. We demonstrate that eosin, which was used to stain the formalin-fixed tissue during processing, yields a discrete 71 base peak of similar morphology to the contaminant peak on CE analysis. The data suggest that eosin in the fixed tissue was not completely eliminated during nucleic acid extraction, resulting in the artifact peak. We discuss the implications of this potentially common contaminant on the interpretation of CE data and demonstrate that artifacts caused by eosin can be avoided by using more stringent DNA purification steps. Histological dyes may fluoresce, and artifacts from them should be considered when primary peaks contain additional underlying peaks of other colors. PMID:15681487

  7. Preparation of denatured sup(99m)Tc labeled HSA aerosols of different median diameters for various imaging studies

    Energy Technology Data Exchange (ETDEWEB)

    Raghunath, B.; Kotrappa, P.; Soni, P.S.; Ganatra, R.D. (Bhabha Atomic Research Centre, Bombay (India))

    1982-02-01

    The preparation of denatured sup(99m)Tc-labelled human serum albumin (HSA) aerosols of different median diameters is described using the BARC (Bhabha Atomic Research Centre) dry aerosol generation and delivery system. The applications of these radioactive aerosols are demonstrated in aerosol scintigraphy of lungs, mucociliary movement studies and lymphoscintigraphy in rabbits. It is concluded that the BARC system gives a simplified, rapid and versatile procedure for generation of denatured volume tagged HSA aerosols for a variety of clinical applications.

  8. Generalized polymer effective charge measurement by capillary isotachophoresis.

    Science.gov (United States)

    Chamieh, Joseph; Koval, Dušan; Besson, Adeline; Kašička, Václav; Cottet, Hervé

    2014-11-28

    In this work, we have generalized the use of capillary isotachophoresis as a universal method for determination of effective charge of anionic and cationic (co)polymers on ordinary capillary electrophoresis instruments. This method is applicable to a broad range of strong or weak polyelectrolytes with good repeatability. Experimental parameters (components and concentrations of leading and terminating electrolytes, capillary diameters, constant electric current intensity) were optimized for implementation in 100 μm i.d. capillaries for both polyanions and polycations. Determined values of polymer effective charge were in a very good agreement with those obtained by capillary electrophoresis with indirect UV detection. Uncertainty of the effective charge measurement using isotachophoresis was addressed and estimated to be ∼5-10% for solutes with mobilities in the 20-50 × 10(-9)m(2)V(-1)s(-1) range. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Capillary electrophoresis: principles and applications in illicit drug analysis.

    Science.gov (United States)

    Tagliaro, F; Turrina, S; Smith, F P

    1996-02-09

    Capillary electrophoresis, which appeared in the early 1980s, is now rapidly expanding into many scientific disciplines, including analytical chemistry, biotechnology and biomedical and pharmaceutical sciences. In capillary electrophoresis,electrokinetic separations are carried out in tiny capillaries at high voltages (10-30 kV), thus obtaining high efficiencies (N > 10(5)) and excellent mass sensitivities (down to 10(-18)-10(-20) moles). The main features of capillary electrophoresis are: versatility of application (from inorganic ions to large DNA fragments), use of different separation modes with different selectivity, extremely low demands on sample volume, negligible running costs, possibility of interfacing with different detection systems, ruggedness and simplicity of instrumentation. Capillary electrophoresis applications in forensic sciences have appeared only recently, but are now rapidly growing, particularly in forensic toxicology. The present paper briefly describes the basic principles of capillary electrophoresis, from both the instrumental and analytical points of view. Furthermore, the main applications in the analysis of illicit/controlled drugs in both illicit preparations and biological samples are presented and discussed (43 references). It is concluded that the particular separation mechanism and the high complementarity of this technique to chromatography makes capillary electrophoresis a new powerful tool of investigation in the hands of forensic toxicologists.

  10. Pulsed-field gel electrophoresis of bacterial chromosomes.

    Science.gov (United States)

    Mawer, Julia S P; Leach, David R F

    2013-01-01

    The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.

  11. Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease.

    Science.gov (United States)

    Rout, Manoj Kumar; Hosur, Ramakrishna V

    2009-02-01

    Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and (15)N relaxation data (R(1), R(2), NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

  12. Generalized polymer effective charge measurement by capillary isotachophoresis

    Czech Academy of Sciences Publication Activity Database

    Chamieh, J.; Koval, Dušan; Besson, A.; Kašička, Václav; Cottet, H.

    2014-01-01

    Roč. 1370, Nov 28 (2014), s. 255-262 ISSN 0021-9673 R&D Projects: GA ČR(CZ) GA13-17224S; GA MŠk 7AMB12FR012 Grant - others:GA AV ČR(CZ) M200551207 Institutional support: RVO:61388963 Keywords : polymer effective charge * polyelectrolyte * isotachophoresis * counter-ion condensation * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.169, year: 2014

  13. Surface Charge Measurement of SonoVue, Definity and Optison: A Comparison of Laser Doppler Electrophoresis and Micro-Electrophoresis.

    Science.gov (United States)

    Ja'afar, Fairuzeta; Leow, Chee Hau; Garbin, Valeria; Sennoga, Charles A; Tang, Meng-Xing; Seddon, John M

    2015-11-01

    Microbubble (MB) contrast-enhanced ultrasonography is a promising tool for targeted molecular imaging. It is important to determine the MB surface charge accurately as it affects the MB interactions with cell membranes. In this article, we report the surface charge measurement of SonoVue, Definity and Optison. We compare the performance of the widely used laser Doppler electrophoresis with an in-house micro-electrophoresis system. By optically tracking MB electrophoretic velocity in a microchannel, we determined the zeta potentials of MB samples. Using micro-electrophoresis, we obtained zeta potential values for SonoVue, Definity and Optison of -28.3, -4.2 and -9.5 mV, with relative standard deviations of 5%, 48% and 8%, respectively. In comparison, laser Doppler electrophoresis gave -8.7, +0.7 and +15.8 mV with relative standard deviations of 330%, 29,000% and 130%, respectively. We found that the reliability of laser Doppler electrophoresis is compromised by MB buoyancy. Micro-electrophoresis determined zeta potential values with a 10-fold improvement in relative standard deviation. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  14. Polymer blends

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Scott D.; Naik, Sanjeev

    2017-08-22

    The present invention provides, among other things, extruded blends of aliphatic polycarbonates and polyolefins. In one aspect, provided blends comprise aliphatic polycarbonates such as poly(propylene carbonate) and a lesser amount of a crystalline or semicrystalline polymer. In certain embodiments, provided blends are characterized in that they exhibit unexpected improvements in their elongation properties. In another aspect, the invention provides methods of making such materials and applications of the materials in applications such as the manufacture of consumer packaging materials.

  15. Topographical properties of polymer films deposited in capillaries for electrophoretic separations of large organic molecules

    Czech Academy of Sciences Publication Activity Database

    Guryča, Vilém; Pacáková, V.; Tlusťáková, Marie; Štulík, K.; Michálek, Jiří

    2004-01-01

    Roč. 27, č. 13 (2004), s. 1121-1129 ISSN 1615-9306 R&D Projects: GA AV ČR IBS4050005; GA AV ČR KSK4055109 Institutional research plan: CEZ:AV0Z4050913 Keywords : capillary electrophoresis * hydrogel polymer coating * column technology Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.927, year: 2004

  16. A study of the thermal denaturation of the S-layer protein from Lactobacillus salivarius

    Science.gov (United States)

    Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian

    2012-09-01

    Surface layer (S-layer) proteins display an intrinsic self-assembly property, forming monomolecular crystalline arrays, identified in outermost structures of the cell envelope in many organisms, such as bacteria and archaea. Isolated S-layer proteins also possess the ability to recrystallize into regular lattices, being used in biotechnological applications, such as controlling the architecture of biomimetic surfaces. To this end, the stability of the S-layer proteins under high-temperature conditions is very important. In this study, the S-layer protein has been isolated from Lactobacillus salivarius 16 strain of human origin, and purified by cation-exchange chromatography. Using circular dichroism (CD) spectroscopy, we have investigated the thermal denaturation of the S-layer protein. The far- and near-UV CD spectra have been collected, and the temperature dependence of the CD signal in these spectral domains has been analyzed. The variable temperature results show that the secondary and tertiary structures of the S-layer protein change irreversibly due to the heating of the sample. After the cooling of the heated protein, the secondary and tertiary structures are partially recovered. The denaturation curves show that the protein unfolding depends on the sample concentration and on the heating rate. The secondary and tertiary structures of the protein suffer changes in the same temperature range. We have also detected an intermediate state in the protein denaturation pathway. Our results on the thermal behavior of the S-layer protein may be important for the use of S-layer proteins in biotechnological applications, as well as for a better understanding of the structure and function of S-layer proteins.

  17. Helical Propensity Affects the Conformational Properties of the Denatured State of Cytochrome c'.

    Science.gov (United States)

    Danielson, Travis A; Bowler, Bruce E

    2018-01-23

    Changing the helical propensity of a polypeptide sequence might be expected to affect the conformational properties of the denatured state of a protein. To test this hypothesis, alanines at positions 83 and 87 near the center of helix 3 of cytochrome c' from Rhodopseudomonas palustris were mutated to serine to decrease the stability of this helix. A set of 13 single histidine variants in the A83S/A87S background were prepared to permit assessment of the conformational properties of the denatured state using histidine-loop formation in 3 M guanidine hydrochloride. The data are compared with previous histidine-heme loop formation data for wild-type cytochrome c'. As expected, destabilization of helix 3 decreases the global stabilities of the histidine variants in the A83S/A87S background relative to the wild-type background. Loop stability versus loop size data yields a scaling exponent of 2.1 ± 0.2, similar to the value of 2.3 ± 0.2 obtained for wild-type cytochrome c'. However, the stabilities of all histidine-heme loops, which contain the helix 3 sequence segment, are increased in the A83S/A87S background compared to the wild-type background. Rate constants for histidine-heme loop breakage are similar for the wild-type and A83S/A87S variants. However, for histidine-heme loops that contain the helix 3 sequence segment, the rate constants for loop formation increase in the A83S/A87S background compared to the wild-type background. Thus, residual helical structure appears to stiffen the polypeptide chain slowing loop formation in the denatured state. The implications of these results for protein folding mechanisms are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Single Molecular Level Probing of Structure and Dynamics of Papain Under Denaturation.

    Science.gov (United States)

    Sengupta, Bhaswati; Chaudhury, Apala; Das, Nilimesh; Sen, Pratik

    2017-01-01

    Papain is a cysteine protease enzyme present in papaya and known to help in digesting peptide. Thus the structure and function of the active site of papain is of interest. The objective of present study is to unveil the overall structural transformation and the local structural change around the active site of papain as a function of chemical denaturant. Papain has been tagged at Cys-25 with a thiol specific fluorescence probe N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA). Guanidine hydrochloride (GnHCl) has been used as the chemical denaturant. Steady state, time-resolved, and single molecular level fluorescence techniques was applied to map the change in the local environment. It is found that papain undergoes a two-step denaturation in the presence of GnHCl. Fluorescence correlation spectroscopic (FCS) data indicate that the size (hydrodynamic diameter) of native papain is ~36.8 Å, which steadily increases to ~53 Å in the presence of 6M GnHCl. FCS study also reveals that the conformational fluctuation time of papain is 6.3 µs in its native state, which decreased to 2.7 µs in the presence of 0.75 M GnHCl. Upon further increase in GnHCl concentration the conformational fluctuation time increase monotonically till 6 M GnHCl, where the time constant is measured as 14 µs. On the other hand, the measurement of ellipticity, hence the helical structure, by circular dichroism spectroscopy is found to be incapable to capture such structural transformation. It is concluded that in the presence of small amount of GnHCl the active site of papain takes up a more compact structure (although the overall size increases) than in the native state, which has been designated as the intermediate state. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation.

    Science.gov (United States)

    Visentin, Jonathan; Guidicelli, Gwendaline; Moreau, Jean-François; Lee, Jar-How; Taupin, Jean-Luc

    2015-07-01

    Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

    Science.gov (United States)

    Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A

    2016-01-01

    Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

    Science.gov (United States)

    Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.

    2005-09-01

    The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

  2. Denaturation of collagen structures and their transformation under the physical and chemical effects

    Science.gov (United States)

    Ivankin, A.; Boldirev, V.; Fadeev, G.; Baburina, M.; Kulikovskii, A.; Vostrikova, N.

    2017-11-01

    The process of denaturation of collagen structures under the influence of physical and chemical factors play an important role in the manufacture of food technology and the production of drugs for medicine and cosmetology. The paper discussed the problem of the combined effects of heat treatment, mechanical dispersion and ultrasonic action on the structural changes of the animal collagen in the presence of weak protonated organic acids. Algorithm combined effects of physical and chemical factors as a result of the formation of the technological properties of products containing collagen has been shown.

  3. Nanometer-Scale Dissection of Chromosomes by Atomic Force Microscopy Combined with Heat-Denaturing Treatment

    Science.gov (United States)

    Tsukamoto, Kazumi; Kuwazaki, Seigo; Yamamoto, Kimiko; Shichiri, Motoharu; Yoshino, Tomoyuki; Ohtani, Toshio; Sugiyama, Shigeru

    2006-03-01

    We have developed a method for dissecting chromosome fragments with a size of a few hundred nanometers by atomic force microscopy (AFM). By using this method, we demonstrated reproducible dissections of silkworm chromosomes in the pachytene phase. The dissected fragments were successfully recovered on the cantilever tips, as confirmed by fluorescent microscopy using fluorescent stained chromosomes. To recover dissected chromosome fragments from a larger chromosome, such as the human metaphase chromosome of a somatic cell, heat denaturation was found to be effective. Further improvements in this method may lead to a novel tool for isolating valuable genes and/or investigating local genome structures in the near future.

  4. Denaturing high-performance liquid chromatography mutation analysis in patients with reduced Protein S levels

    DEFF Research Database (Denmark)

    Bathum, Lise; Münster, Anna-Marie; Nybo, Mads

    2008-01-01

    diagnosis and risk estimation. The aim was to design a high-throughput genetic analysis based on denaturing high-performance liquid chromatography to identify sequence variations in the gene coding for Protein S. PATIENTS: In total, 55 patients referred to the Section of Thrombosis and Haemostasis, Odense......BACKGROUND: Patients with congenital Protein S deficiency have increased risk of venous thromboembolism. However, Protein S levels show large intra-individual variation and the biochemical assays have low accuracy and a high interlaboratory variability. Genetic analysis might aid in a more precise......, giving a precise diagnosis and subsequently a better risk estimation....

  5. DNA denaturation through a model of the partition points on a one-dimensional lattice

    International Nuclear Information System (INIS)

    Mejdani, R.; Huseini, H.

    1994-08-01

    We have shown that by using a model of the partition points gas on a one-dimensional lattice, we can study, besides the saturation curves obtained before for the enzyme kinetics, also the denaturation process, i.e. the breaking of the hydrogen bonds connecting the two strands, under treatment by heat of DNA. We think that this model, as a very simple model and mathematically transparent, can be advantageous for pedagogic goals or other theoretical investigations in chemistry or modern biology. (author). 29 refs, 4 figs

  6. The expression of miR-125b regulates angiogenesis during the recovery of heat-denatured HUVECs.

    Science.gov (United States)

    Zhou, Situo; Zhang, Pihong; Liang, Pengfei; Huang, Xiaoyuan

    2015-06-01

    In previous studies we found that miR-125b was down-regulated in denatured dermis of deep partial thickness burn patients. Moreover, miR-125b inhibited tumor-angiogenesis associated with the decrease of ERBB2 and VEGF expression in ovarian cancer cells and breast cancer cells, etc. In this study, we investigated the expression patterns and roles of miR-125b during the recovery of denatured dermis and heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burns in Sprague-Dawley rats and the heat-denatured cells (52°C, 35 s) were used for analysis. Western blot analysis and real-time PCR were applied to evaluate the expression of miR-125b and ERBB2 and VEGF. The ability of angiogenesis in heat-denatured HUVECs was analyzed by scratch wound healing and tube formation assay after pri-miR-125b or anti-miR-125b transfection. miR-125b expression was time-dependent during the recovery of heat-denatured dermis and HUVECs. Moreover, miR-125b regulated ERBB2 mRNA and Protein Expression and regulated angiogenesis association with regulating the expression of VEGF in heat-denatured HUVECs. Taken together our results show that the expression of miR-125b is time-dependent and miR-125b plays a regulatory role of angiogenesis during wound healing after burns. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

  7. Dynamic computer simulations of electrophoresis: three decades of active research.

    Science.gov (United States)

    Thormann, Wolfgang; Caslavska, Jitka; Breadmore, Michael C; Mosher, Richard A

    2009-06-01

    Dynamic models for electrophoresis are based upon model equations derived from the transport concepts in solution together with user-inputted conditions. They are able to predict theoretically the movement of ions and are as such the most versatile tool to explore the fundamentals of electrokinetic separations. Since its inception three decades ago, the state of dynamic computer simulation software and its use has progressed significantly and Electrophoresis played a pivotal role in that endeavor as a large proportion of the fundamental and application papers were published in this periodical. Software is available that simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. This has been employed to show the detailed mechanisms of many of the fundamental phenomena that occur in electrophoretic separations. Dynamic electrophoretic simulations are relevant for separations on any scale and instrumental format, including free-fluid preparative, gel, capillary and chip electrophoresis. This review includes a historical overview, a survey of current simulators, simulation examples and a discussion of the applications and achievements of dynamic simulation.

  8. Natural polymers: an overview

    CSIR Research Space (South Africa)

    John, MJ

    2012-08-01

    Full Text Available The scarcity of natural polymers during the world war years led to the development of synthetic polymers like nylon, acrylic, neoprene, styrene-butadiene rubber (SBR) and polyethylene. The increasing popularity of synthetic polymers is partly due...

  9. Capillary array electrophoresis using laser-excited confocal fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Huang, X.C.; Quesada, M.A.; Mathies, R.A. [Univ. of California, Berkeley, CA (United States)

    1992-04-15

    Capillary electrophoresis (CE) has found widespread application in analytical and biomedical research, and the scope and sophistication of CE is still rapidly advancing. Gel-filled capillaries have been employed for the rapid separation and analysis of synthetic polynucleotides, DNA sequencing fragments, and DNA restriction fragments. Open-tube capillary electrophoresis has attained subattomole detection levels in amino acid separations 14 and proven its utility for the separation of proteins, viruses, and bacteria. Separation of the optical isomers of dansyl amino acids has also been successfully demonstrated. Micellar electrokinetic capillary chromatography, isoelectric focusing, and on-column derivatization can all be performed on CE columns, demonstrating the utility of capillary electrophoresis as an analytical and micropreparative tool. 29 refs., 6 figs., 1 tab.

  10. Magneto-paper electrophoresis in the separation of inorganic ions

    International Nuclear Information System (INIS)

    Mukherjee, H.G.; Datta, S.K.

    1983-01-01

    A comparative study of the separation of lanthanide ions by paper electrophoresis and magneto-paper electrophoresis is reported. The separation of La(III)-Gd(III), La(III)-Dy(III), Lu(III)-Gd(III), Lu(III)-Ho(III) etc. was achieved by magneto paper electrophoresis using 0.1M KCl as carrier electrolyte. Separation of different oxidation states of the same element like Cu(I)-Cu(II), Ce(III)-Ce(IV), Mn(CN) 6 3 - -Mn(CN) 6 4 - , Co(C 2 O 4 ) 2 2 - -Co(C 2 O 4 ) 3 3 - , V(CN) 6 3 - -VO(CN) 5 3 - , W(CN) 8 4 - -W(CN) 8 3 - and Ru(CN) 6 3 - Ru(CN) 6 4 - was also achieved by magneto paper electrophoretic technique using different carrier electrolytes. (Author)

  11. Electrophoresis for the analysis of heparin purity and quality.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J

    2012-06-01

    The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Detection of AGEs as markers for carbohydrate metabolism and protein denaturation.

    Science.gov (United States)

    Nagai, Ryoji; Shirakawa, Jun-Ichi; Fujiwara, Yukio; Ohno, Rei-Ichi; Moroishi, Narumi; Sakata, Noriyuki; Nagai, Mime

    2014-07-01

    Approximately 100 years have passed since the Maillard reaction was first reported in the field of food chemistry as a condensation reaction between reducing sugars and amino acids. This reaction is thought to progress slowly primarily from glucose with proteins in vivo. An early-stage product, called the "Amadori product", is converted into advanced glycation end products. Those accumulate in the body in accordance with age, with such accumulation being enhanced by lifestyle-related diseases that result in the denaturation of proteins. Recent studies have demonstrated that intermediate carbonyls are generated by several pathways, and rapidly generate many glycation products. However, accurate quantification of glycation products in vivo is difficult due to instability and differences in physicochemical properties. In this connection, little is known about the relationship between the structure of glycation products and pathology. Furthermore, the interaction between proteins modified by glycation and receptors for advanced glycation end products is also known to induce the production of several inflammatory cytokines. Therefore, those inhibitors have been developed over the world to prevent lifestyle-related diseases. In this review, we describe the process of protein denaturation induced by glycation and discuss the possibility of using the process as a marker of age-related diseases.

  13. Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation.

    Science.gov (United States)

    Mikulecky, Peter J; Feig, Andrew L

    2004-01-01

    In proteins, empirical correlations have shown that changes in heat capacity (DeltaC(P)) scale linearly with the hydrophobic surface area buried upon folding. The influence of DeltaC(P) on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to DeltaC(P)s of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a DeltaC(P) of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated DeltaC(P) by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the DeltaC(P). We weigh the strengths and limitations of this method for determining DeltaC(P) values. Finally, we discuss the data in light of the physical origins of the DeltaC(P)s for RNA folding and consider their impact on biological function.

  14. Thermal and chemical denaturation of Bacillus circulans xylanase: A biophysical chemistry laboratory module.

    Science.gov (United States)

    Raabe, Richard; Gentile, Lisa

    2008-11-01

    A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the traditional two-semester biochemistry series. The latter is the model used for biochemistry and molecular biology majors at the University of Richmond, whose second semester of biochemistry is a course entitled Proteins: Structure, Function, and Biophysics. What is described herein is a protein thermodynamics laboratory module, using the protein Bacillus circulans xylanase, which reinforces many lecture concepts, including: (i) the denatured (D) state ensemble of a protein can be different, depending on how it was populated; (ii) intermediate states may be detected by some spectroscopic techniques but not by others; (iii) the use and assumptions of the van't Hoff approach to calculate ΔH(o) , ΔS(o) , and ΔG(o) (T) for thermal protein unfolding transitions; and (iv) the use and assumptions of an approach that allows determination of the Gibb's free energy of a protein unfolding transition based on the linear dependence of ΔG(o) on the concentration of denaturant used. This module also requires students to design their own experimental protocols and spend time in the primary literature, both important parts of an upper division lab. Copyright © 2008 International Union of Biochemistry and Molecular Biology, Inc.

  15. Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

    Science.gov (United States)

    Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

    2017-05-15

    Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Effects of thermally induced denaturation on technological-functional properties of whey protein isolate-based films.

    Science.gov (United States)

    Schmid, M; Krimmel, B; Grupa, U; Noller, K

    2014-09-01

    This study examined how and to what extent the degree of denaturation affected the technological-functional properties of whey protein isolate (WPI)-based coatings. It was observed that denaturation affected the material properties of WPI-coated films significantly. Surface energy decreased by approximately 20% compared with native coatings. Because the surface energy of a coating should be lower than that of the substrate, this might result in enhanced wettability characteristics between WPI-based solution and substrate surface. Water vapor barrier properties increased by about 35% and oxygen barrier properties increased by approximately 33%. However, significant differences were mainly observed between coatings made of fully native WPI and ones with a degree of denaturation of 25%. Higher degrees of denaturation did not lead to further improvement of material properties. This observation offers cost-saving potential: a major share of denatured whey proteins may be replaced by fully native ones that are not exposed to energy-intensive heat treatment. Furthermore, native WPI solutions can be produced with higher dry matter content without gelatinizing. Hence, less moisture has to be removed through drying, resulting in reduced energy consumption. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  17. Ion-ion interactions in the denatured state contribute to the stabilization of CutA1 proteins.

    Science.gov (United States)

    Yutani, Katsuhide; Matsuura, Yoshinori; Naitow, Hisashi; Joti, Yasumasa

    2018-05-16

    In order to elucidate features of the denatured state ensembles that exist in equilibrium with the native state under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 K using the monomer subunits of three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) with denaturation temperature (T d ) = 85.6 °C, a hydrophobic mutant (Ec0VV) with T d  = 113.3 °C, and an ionic mutant (Ec0VV_6) with T d  = 136.8 °C. The occupancy of salt bridges by the six substituted charged residues in Ec0VV_6 was 140.1% at 300 K and 89.5% at 450 K, indicating that even in the denatured state, salt bridge occupancy was high, approximately 60% of that at 300 K. From these results, we can infer that proteins from hyperthermophiles with a high ratio of charged residues are stabilized by a decrease in conformational entropy due to ion-ion interactions in the denatured state. The mechanism must be comparable to the stabilization conferred by disulfide bonds within a protein. This suggests that introduction of charged residues, to promote formation of salt bridges in the denatured state, would be a simple way to rationally design stability-enhanced mutants.

  18. Capillary electrophoresis with indirect UV detection for the determination of stabilizers and citrates present in human albumin solutions.

    Science.gov (United States)

    Jaworska, Małgorzata; Cygan, Paulina; Wilk, Małgorzata; Anuszewska, Elzbieta

    2009-08-15

    Sodium caprylate and N-acetyltryptophan are the most frequently used stabilizers that protect the albumin from aggregation or heat induced denaturation. In turn citrates - excipients remaining after fractionation process - can be treated as by-product favoring leaching aluminum out of glass containers whilst albumin solution is stored. With ionic nature these substances have all the markings of a subject for capillary electrophoresis analysis. Thus CE methods were proposed as new approach for quality control of human albumin solution in terms of determination of stabilizers and citrates residue. Human albumin solutions both 5% and 20% from various manufacturers were tested. Indirect detection mode was set to provide sufficient detectability of analytes lacking of chromophores. As being anions analytes were separated with reversed electroosmotic flow. As a result of method optimization two background electrolytes based on p-hydroxybenzoic acid and 2,6-pyridinedicarboxylic acid were selected for stabilizers and citrates separation, respectively. The optimized methods were successfully validated. For citrates that require quantification below 100microM the method demonstrated the precision less than 4% and the limit of detection at 4microM. In order to check the new methods accuracy and applicability the samples were additionally tested with selected reference methods. The proposed methods allow reliable quantification of stabilizers and citrates in human albumin solution that was confirmed by method validation as well as result comparison with reference methods. The CE methods are considered to be suitable for quality control yet simplifying and reducing cost of analysis.

  19. Images of gel electrophoresis - RGP caps | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us RGP caps Images of gel electrophoresis Data detail Data name Images of gel electrophoresis D...OI 10.18908/lsdba.nbdc00318-05-002 Description of data contents Detailed information and images of gel electrophoresis... of each marker. Data file File name: rgp_caps_electrophoresis_image.zip File URL: ftp://ftp.biosc...iencedbc.jp/archive/rgp-caps/LATEST/rgp_caps_electrophoresis_image.zip File size:... 28.7 MB Simple search URL - Data acquisition method Gel electrophoresis Data analysis method STS markers :

  20. Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

    Science.gov (United States)

    WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

    2013-01-01

    Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

  1. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    Science.gov (United States)

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  2. Polymer dynamics from synthetic polymers to proteins

    Indian Academy of Sciences (India)

    Keywords. Polymer dynamics; reptation; domain dynamics biomolecules. Abstract. Starting from the standard model of polymer motion - the Rouse model - we briefly present some key experimental results on the mesoscopic dynamics of polymer systems. We touch the role of topological confinement as expressed in the ...

  3. A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

    International Nuclear Information System (INIS)

    Castronuovo, Giuseppina; Niccoli, Marcella

    2012-01-01

    Highlights: ► A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. ► The enthalpic interaction coefficients are very useful parameters to gain information about the mechanism through which two hydrated molecules interact in solution. ► Hypotheses are proposed about the mechanism underlying the denaturation of lysozyme induced by high concentrations of urea or ethanol. - Abstract: A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg −1 , the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules. The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on the possible mode of action of the two cosolvents on the stability of proteins

  4. A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

    Energy Technology Data Exchange (ETDEWEB)

    Castronuovo, Giuseppina, E-mail: giuseppina.castronuovo@unina.it [Department of Chemistry, University Federico II of Naples, Complesso Universitario Monte S. Angelo, via Cintia, 80126 Naples (Italy); Niccoli, Marcella [Department of Chemistry, University Federico II of Naples, Complesso Universitario Monte S. Angelo, via Cintia, 80126 Naples (Italy)

    2012-09-10

    Highlights: Black-Right-Pointing-Pointer A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Black-Right-Pointing-Pointer The enthalpic interaction coefficients are very useful parameters to gain information about the mechanism through which two hydrated molecules interact in solution. Black-Right-Pointing-Pointer Hypotheses are proposed about the mechanism underlying the denaturation of lysozyme induced by high concentrations of urea or ethanol. - Abstract: A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg{sup -1}, the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules. The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on

  5. Chemical denaturation of globular proteins at the air/water interface: an x-ray and neutron reflectometry study

    International Nuclear Information System (INIS)

    Perriman, A.W.; Henderson, M.J.; White, J.W.

    2003-01-01

    Full text: X-ray and neutron reflectometry has been used to probe the equilibrium surface structure of hen egg white lysozyme (lysozyme) and bovine β -lactoglobulin (β -lactoglobulin) under denaturing conditions at the air-water interface. This was achieved by performing experiments on 10 mg mL -1 protein solutions containing increasing concentrations of the chemical denaturant guanidinium hydrochloride (G.HCl). For solutions containing no G.HCl, the surface structure of the proteins was represented by a two-layer model with total thicknesses of 48 Angstroms and 38 Angstroms for lysozyme and β -lactoglobulin, respectively. The total volume of a single protein molecule and the associated water molecules was evaluated to be approximately 45 (0.3) nm 3 for lysozyme, and 60 (0.3) nm 3 for β-lactoglobulin. The thickness dimensions and the total volumes compared favourably with the crystal dimensions of 45 x 30 x 30 Angstroms (40.5 nm 3 ),1 and 36 x 36 x 36 Angstroms (47 nm 3 ) 2 for lysozyme and β -lactoglobulin, respectively. This comparison suggests that when no denaturant was present, the structures of lysozyme and β -lactoglobulin were near to their native conformations at the air-water interface. The response to the presence of the chemical denaturant was different for each protein. The surface layer of β-lactoglobulin expanded at very low concentrations (0.2 mol dm -3 ) of G.HCl. In contrast, the lysozyme layer contracted. At higher concentrations, unfolding of both the proteins led to the formation of a third diffuse layer. In general, lysozyme appeared to be less responsive to the chemical denaturant, which is most likely a result of the higher disulfide content of lysozyme. A protocol allowing quantitative thermodynamic analysis of the contribution from the air-water interface to the chemical denaturation of a protein was developed

  6. Biological function evaluation and effects of laser micro-pore burn-denatured acellular dermal matrix.

    Science.gov (United States)

    Zhang, Youlai; Zeng, Yuanlin; Xin, Guohua; Zou, Lijin; Ding, Yuewei; Duyin, Jiang

    2018-03-01

    In the field of burns repairs, many problems exist in the shortage of donor skin, the expense of allograft or xenograft skin, temporary substitution and unsatisfactory extremity function after wound healing. Previous studies showed that burn-denatured skin could return to normal dermis formation and function. This study investigates the application of laser micro-pore burn-denatured acellular dermis matrix (DADM) from an escharotomy in the repair of burn wounds and evaluates the biological properties and wound repair effects of DADM in implantation experiments in Kunming mice. Specific-pathogen-free (SPF) Kunming mice were used in this study. A deep II° burn wound was created on the dorsum of the mice by an electric heated water bath. The full-thickness wound tissue was harvested. The necrotic tissue and subcutaneous tissue were removed. The denatured dermis was preserved and treated with 0.25% trypsin, 0.5% Triton X-100. The DADM was drilled by laser micro-pore. The biological properties and grafting effects of laser micro-pore burn-DADM were evaluated by morphology, cytokine expression levels and subcutaneous implantation experiments in Kunming mice. We found statistical significance (Ppore burn-DADM (experimental group) compared to the control group (no laser micro-pore burn-DADM). Cytokine expression level was different in the dermal matrixes harvested at various time points after burn (24h, 48h, 72h and infected wound group). Comparing the dermal matrix from 24h burn tissue to infected wound tissue, the expression level of IL-6, MMP-24, VE-cadherin and VEGF were decreased. We found no inflammatory cells infiltration in the dermal matrix were observed in both experimental and control groups (24h burn group), while the obviously vascular infiltration and fiber fusion were observed in the experimental group after subcutaneous implantation experiments. There was better bio-performance, low immunogenicity and better dermal incorporation after treated by laser

  7. Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

    Science.gov (United States)

    Anumalla, Bramhini; Prabhu, N Prakash

    2018-01-25

    When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two

  8. Preparation of denatured sup(99m)Tc labeled HSA aerosols of different median diameters for various imaging studies

    International Nuclear Information System (INIS)

    Raghunath, B.; Kotrappa, P.; Soni, P.S.; Ganatra, R.D.

    1982-01-01

    The preparation of denatured sup(99m)Tc-labelled human serum albumin (HSA) aerosols of different median diameters is described using the BARC (Bhabha Atomic Research Centre) dry aerosol generation and delivery system. The applications of these radioactive aerosols are demonstrated in aerosol scintigraphy of lungs, mucociliary movement studies and lymphoscintigraphy in rabbits. It is concluded that the BARC system gives a simplified, rapid and versatile procedure for generation of denatured volume tagged HSA aerosols for a variety of clinical applications. (U.K.)

  9. Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation.

    Science.gov (United States)

    Singh, Upendra Kumar; Patel, Rajan

    2018-05-25

    In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C 8 mim][Cl], and 1-decyl-3-methylimidazolium chloride [C 10 mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C 8 mim] + and [C 10 mim] + cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C 10 mim] + cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C 8 mim] + cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C 10 mim][Cl], and this was correlated with the fast and medium lifetimes (τ 1 and τ 2 ) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C 10 mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C 10 mim] + cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.

  10. 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

  11. 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

  12. Application of a diode-array detector in capillary electrophoresis

    NARCIS (Netherlands)

    Beck, W.; Hoek, van R.; Engelhardt, H.

    1993-01-01

    In the last decade diode-array detection has proved to be extremely useful in high performance liquid chromatography in recording UV-visible spectra directly and on-line in the column effluent. In capillary electrophoresis (CE) only fast-scanning detectors with long scan times (up to 2 s) are

  13. Defining carbohydrate binding of glucan phosphatases via Affinity gel electrophoresis

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2016-01-01

    was to determine a technique to measure carbohydrate binding quickly and efficiently. We established a protocol to reproducibly and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE). The results show that the various glucan phosphatases possess differing...

  14. Thermostatted dual-channel portable capillary electrophoresis instrument

    Czech Academy of Sciences Publication Activity Database

    Koenka, I.J.; Küng, N.; Kubáň, Pavel; Chwalek, T.; Furrer, G.; Wehrli, B.; Müller, B.; Hauser, P.C.

    2016-01-01

    Roč. 37, 17-18 (2016), s. 2368-2375 ISSN 0173-0835 Institutional support: RVO:68081715 Keywords : portable devices * on-site measurements * capillary electrophoresis Subject RIV: CB - Analytical Chemistry , Separation Impact factor: 2.744, year: 2016

  15. Gel Electrophoresis and Fluorescamine Methods for the Detection of ...

    African Journals Online (AJOL)

    For the fluorescamine method, clarification was achieved by isoelectric precipitation and precipitation with acid to obtain pH 4.6 and 6% TCA soluble extracts respectively. Non-clarified samples were used for gel electrophoresis. Both methods confirmed that raw milk and milk processed at 85/15s were the most proteolysed, ...

  16. Characterization of metal/humic acid systems by Capillary Electrophoresis

    NARCIS (Netherlands)

    Staden JJ van; Hoop MAGT van den; Cleven R; LAC

    2000-01-01

    Metal-humic acid systems have been characterised applying Capillary Electrophoresis (CE). Appropriate experimental conditions with respect to carrier electrolyte, pH range, salt concentration, humic acid concentration and the applied potential, have been optimised. The influence of multivalent metal

  17. Strain identification in Rhizobium by starch gel electrophoresis of isoenzymes

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen; Nielsen, G.

    1985-01-01

    Sonieated extracts of rhizobia, especiaUy Rhizobium leguminosarum from pea and vetch, were run in horizontal starch gel electrophoresis in the cold. The rhizobia were grown on agar on a slime suppressing substrate of tryptone-yeast extract-CaCl2 with small amounts of mannitol, sorbitol...

  18. Capillary electrophoresis in the analysis of biologically important thiols

    Czech Academy of Sciences Publication Activity Database

    Lačná, J.; Kubáň, Petr; Foret, František

    2017-01-01

    Roč. 38, č. 1 (2017), s. 203-222 ISSN 0173-0835 Institutional support: RVO:68081715 Keywords : biological thiols * capillary electrophoresis * clinical applications Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 2.744, year: 2016

  19. Thermostatted dual-channel portable capillary electrophoresis instrument

    Czech Academy of Sciences Publication Activity Database

    Koenka, I.J.; Küng, N.; Kubáň, Pavel; Chwalek, T.; Furrer, G.; Wehrli, B.; Müller, B.; Hauser, P.C.

    2016-01-01

    Roč. 37, 17-18 (2016), s. 2368-2375 ISSN 0173-0835 Institutional support: RVO:68081715 Keywords : portable devices * on-site measurements * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.744, year: 2016

  20. Gel Electrophoresis--The Easy Way for Students

    Science.gov (United States)

    VanRooy, Wilhelmina; Sultana, Khalida

    2010-01-01

    This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

  1. Device for Horizontal Zone Electrophoresis in Free Electrolyte

    CERN Document Server

    Priemyshev, A N; Bozhikov, G A; Alikov, B A; Salamatin, A V; Furyaev, T A; Maslov, O D; Milanov, M V; Dmitriev, S N

    2000-01-01

    With expansion of area of application of an electromigration method the necessity of modernization of installation for horizontal zone electrophoresis in free electrolyte has appeared. A number of the basic modules was essentially advanced, that has allowed considerably increase reliability and accuracy of received results. The device is completely automated.

  2. Analysis of Two-Dimensional Electrophoresis Gel Images

    DEFF Research Database (Denmark)

    Pedersen, Lars

    2002-01-01

    This thesis describes and proposes solutions to some of the currently most important problems in pattern recognition and image analysis of two-dimensional gel electrophoresis (2DGE) images. 2DGE is the leading technique to separate individual proteins in biological samples with many biological...

  3. Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates

    Science.gov (United States)

    Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by th...

  4. Determination of propionate in bread using capillary zone electrophoresis

    NARCIS (Netherlands)

    Ackermans, M.T.; Ackermans-Loonen, J.C.J.M; Beckers, J.L.

    1992-01-01

    A method for the determination of propionate in bread is described. The propionate was extracted from the bread with a repeated extraction procedure and measured using capillary zone electrophoresis in the indirect UV mode applying a background electrolyte of 0.005 M Tris adjusted at pH 4.6 by

  5. Evaluation of wheat by polyacrylamide gel electrophoresis | Shuaib ...

    African Journals Online (AJOL)

    ... polyacrylamide gel electrophoresis (SDS-PAGE). Electrophorogram for each variety were scored and presence or absence of each band noted and was entered in a binary data matrix. Based on the data of SDS-PAGE gels cluster analysis was performed to check the variations among varieties. The overall result shows ...

  6. Two-dimensional gel electrophoresis analysis of different parts of ...

    African Journals Online (AJOL)

    Two-dimensional gel electrophoresis analysis of different parts of Panax quinquefolius L. root. ... From these results it was concluded that proteomic analysis method was an effective way to identify the different parts of quinquefolius L. root. These findings may contribute to further understanding of the physiological ...

  7. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a significant increase in appearance of apoptosis when using single cell gel electrophoresis assay. The present report demonstrates that the characteristic pattern of apoptotic comets detected by the comet assay ...

  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...

  9. Routine hemoglobin electrophoresis for pediatric surgery day case ...

    African Journals Online (AJOL)

    Background: Hemoglobin electrophoresis (HBE) is a part of the preoperative routine requested by anesthetists. However, the prevalence of hemoglobinopathy in the population is low. This study aims to determine the clinical risk factors for hemoglobinopathies and propose clinical guidelines for preoperative screening of ...

  10. A Study on Major Components of Bee Venom Using Electrophoresis

    Directory of Open Access Journals (Sweden)

    Lee, Jin-Seon

    2000-12-01

    Full Text Available This study was designed to study on major components of various Bee Venom(Bee Venom by electrical stimulation in Korea; K-BV I, Bee Venom by Microwave stimulation in Korea; K -BV II, 0.5rng/ml, Fu Yu Pharmaceutical Factory, China; C-BV, 1mg /ml, Monmouth Pain Institute, Inc., U.S.A.; A-BV using Electrophoresis. The results were summarized as follows: 1. In 1:4000 Bee Venom solution rate, the band was not displayed distinctly usmg Electrophoresis. But in 1: 1000, the band showed clearly. 2. The results of Electrophoresis at solution rate 1:1000, K-BV I and K-BVII showed similar band. 3. The molecular weight of Phospholipase A2 was known as 19,000 but its band was seen at 17,000 in Electrophoresis. 4. Protein concentration of Bee Venom by Lowry method was different at solution rate 1:4000 ; C-BV was 250μg/ml, K-BV I was 190μg/ml, K-BV Ⅱ was 160μg/ml and C-BV was 45μg/ml. 5. Electrophoresis method was unuseful for analysis of Bee Venom when solution rate is above 1:4000 but Protein concentration of Bee Venom by Lowry method was possible. These data from the study can be applied to establish the standard measurement of Bee Venom and prevent pure bee venom from mixing of another components. I think it is desirable to study more about safety of Bee Venom as time goes by.

  11. Fuel utilization improvement in PWRs using the denatured 233U-Th cycle

    International Nuclear Information System (INIS)

    Jones, H.M.; Schwenk, G.A.; Toops, E.C.; Yotinen, V.O.

    1980-06-01

    A number of changes in PWR core design and/or operating strategy were evaluated to assess the fuel utilization improvement achievable by their implementation in a PWR using thorium-based fuel and operating in a recycle mode. The reference PWR for this study was identical to the B and W Standard Plant except that the fuel pellets were of denatured ( 233 U/ 238 U-Th)O 2 . An initial scoping study identified the three most promising improvement concepts as (1) a very tight lattice, (2) thorium blankets, and (3) ThO 2 rods placed in available guide tubes. A conceptual core design incorporating these changes was then developed, and the fuel utilization of this modified design was compared with that of the reference case

  12. Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

    Science.gov (United States)

    Sinha, Rajeshwari; Khare, Sunil K.

    2014-01-01

    Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants. PMID:24782853

  13. Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

    Science.gov (United States)

    Nemtseva, Elena V; Lashchuk, Olesya O; Gerasimova, Marina A; Melnik, Tatiana N; Nagibina, Galina S; Melnik, Bogdan S

    2017-12-21

    In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

  14. 125I-labeled cortisol radioimmunoassay in which serum binding protein are enzymatically denatured

    International Nuclear Information System (INIS)

    Hasler, M.J.; Painter, K.; Niswender, G.D.

    1976-01-01

    We report an iodine-125 radioimmunoassay for cortisol in biological fluids, in which interfering binding proteins are enzymatically denatured. An antiserum to cortisol-3-carboxymethyloxime-bovine serum albumin, extremely low cross-reacting with other corticosteroids, was raised in rabbits. A cortisol-3-carboxymethyloxime tyrosine methyl ester derivative was synthesized and labeled with iodine-125 by standard radioiodination techniques. To eliminate the need for extraction and recovery procedures, we digested interfering binding with a proteolytic enzyme, which then was heat-inactivated before adding the labeled derivative and the premixed, preincubated antiserum complex. There was quantitative analytical recovery of esogenous cortisol added to sera from a normal man, a normal woman, and a pregnant woman. Values for the same samples agreed after extraction and chromatographic purification and agreed well with values obtained by other techniques by independent reference laboratories. The five-step assay can be done in 6 h or less

  15. Continuous Fractionation of a two-component mixture by zone electrophoresis

    NARCIS (Netherlands)

    Zalewski, D.R.; Gardeniers, Johannes G.E.

    2009-01-01

    Synchronized continuous-flow zone electrophoresis is a recently demonstrated tool for performing electrophoretic fractionation of a complex sample. The method resembles free flow electrophoresis, but unlike in that technique, no mechanical fluid pumping is required. Instead, fast electrokinetic flow

  16. Thermal, chemical and pH induced unfolding of turmeric root lectin: modes of denaturation.

    Directory of Open Access Journals (Sweden)

    Himadri Biswas

    Full Text Available Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol-1 and 14.90 Kcal mol-1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp is 3.42 Kcal mol-1 K-1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.

  17. Aqueous Solutions of the Ionic Liquid 1-butyl-3-methylimidazolium Chloride Denature Proteins

    International Nuclear Information System (INIS)

    Baker, Gary A.; Heller, William T.

    2009-01-01

    As we advance our understanding, ionic liquids (ILs) are finding ever broader scope within the chemical sciences including, most recently, pharmaceutical, enzymatic, and bioanalytical applications. With examples of enzymatic activity reported in both neat ILs and in IL/water mixtures, enzymes are frequently assumed to adopt a quasi-native conformation, even if little work has been carried out to date toward characterizing the conformation, dynamics, active-site perturbation, cooperativity of unfolding transitions, free energy of stabilization, or aggregation/oligomerization state of enzymes in the presence of an IL solvent component. In this study, human serum albumin and equine heart cytochrome c were characterized in aqueous solutions of the fully water-miscible IL 1-butyl-3-methylimidazolium chloride, (bmim)Cl, by small-angle neutron and X-ray scattering. At (bmim)Cl concentrations up to 25 vol.%, these two proteins were found to largely retain their higher-order structures whereas both proteins become highly denatured at the highest IL concentration studied here (i.e., 50 vol.% (bmim)Cl). The response of these proteins to (bmim)Cl is analogous to their behavior in the widely studied denaturants guanidine hydrochloride and urea which similarly lead to random coil conformations at excessive molar concentrations. Interestingly, human serum albumin dimerizes in response to (bmim)Cl, whereas cytochrome c remains predominantly in monomeric form. These results have important implications for enzymatic studies in aqueous IL media, as they suggest a facile pathway through which biocatalytic activity can be altered in these nascent and potentially green electrolyte systems

  18. Shape memory polymers

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Thomas S.; Bearinger, Jane P.

    2017-08-29

    New shape memory polymer compositions, methods for synthesizing new shape memory polymers, and apparatus comprising an actuator and a shape memory polymer wherein the shape memory polymer comprises at least a portion of the actuator. A shape memory polymer comprising a polymer composition which physically forms a network structure wherein the polymer composition has shape-memory behavior and can be formed into a permanent primary shape, re-formed into a stable secondary shape, and controllably actuated to recover the permanent primary shape. Polymers have optimal aliphatic network structures due to minimization of dangling chains by using monomers that are symmetrical and that have matching amine and hydroxl groups providing polymers and polymer foams with clarity, tight (narrow temperature range) single transitions, and high shape recovery and recovery force that are especially useful for implanting in the human body.

  19. Shape memory polymers

    Science.gov (United States)

    Wilson, Thomas S.; Bearinger, Jane P.

    2015-06-09

    New shape memory polymer compositions, methods for synthesizing new shape memory polymers, and apparatus comprising an actuator and a shape memory polymer wherein the shape memory polymer comprises at least a portion of the actuator. A shape memory polymer comprising a polymer composition which physically forms a network structure wherein the polymer composition has shape-memory behavior and can be formed into a permanent primary shape, re-formed into a stable secondary shape, and controllably actuated to recover the permanent primary shape. Polymers have optimal aliphatic network structures due to minimization of dangling chains by using monomers that are symmetrical and that have matching amine and hydroxyl groups providing polymers and polymer foams with clarity, tight (narrow temperature range) single transitions, and high shape recovery and recovery force that are especially useful for implanting in the human body.

  20. 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...

  1. [The sequential use of local vacuum magnetotherapy and papaverine electrophoresis with sinusoidal modulated currents in impotence].

    Science.gov (United States)

    Karpukhin, I V; Bogomol'nyĭ, V A

    1997-01-01

    105 patients with chronic nonspecific prostatitis were examined and treated with papaverin electrophoresis using sinusoidal modulated currents (SMC) and local vacuum magnetotherapy (LVMT). Papaverin SMC electrophoresis and LVMT stimulated cavernous circulation. The highest stimulation was achieved at successive use of LVMT and the electrophoresis. LVMT followed by the electrophoresis maintained good cavernous circulation for 5-6 hours after the procedure in the course of which several spontaneous erections were observed.

  2. T1ρ is superior to T2 mapping for the evaluation of articular cartilage denaturalization with osteoarthritis: radiological-pathological correlation after total knee arthroplasty.

    Science.gov (United States)

    Takayama, Yukihisa; Hatakenaka, Masamitsu; Tsushima, Hidetoshi; Okazaki, Ken; Yoshiura, Takashi; Yonezawa, Masato; Nishikawa, Kei; Iwamoto, Yukihide; Honda, Hiroshi

    2013-04-01

    We compared the diagnostic performance of T1ρ and T2 mappings in the evaluation of denatured articular cartilage with osteoarthritis of the knee. 2D-Sagittal T1ρ and T2 mappings of the knee were obtained from 16 patients before total knee arthroplasty. After surgery, specimens of the femur and tibia were regionally segmented according to a 5-point scale of the severity of denaturalization. The T1ρ and T2 values in the full thickness of the articular cartilage in each region were measured by two observers. The two mappings were compared for their ability to differentiate between normal and denatured articular cartilage and also for their usefulness in grading the severity of the denaturalization using the area under receiver operating characteristic curves (Az). A pT2 mapping for the differentiation between normal and denatured articular cartilage (pT2 mapping could not. However, there were no significant differences between the two mappings in the discrimination of mild versus moderate denaturalization or of moderate versus severe denaturalization. The two observers showed good agreement in the results (intraclass correlation coefficient=0.81 for T1ρ and 0.92 for T2). T1ρ mapping is superior to T2 mapping for the evaluation of denatured articular cartilage with osteoarthritis of the knee. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Polymer nanocomposites: polymer and particle dynamics

    KAUST Repository

    Kim, Daniel

    2012-01-01

    Polymer nanocomposites containing nanoparticles smaller than the random coil size of their host polymer chains are known to exhibit unique properties, such as lower viscosity and glass transition temperature relative to the neat polymer melt. It has been hypothesized that these unusual properties result from fast diffusion of the nanostructures in the host polymer, which facilitates polymer chain relaxation by constraint release and other processes. In this study, the effects of addition of sterically stabilized inorganic nanoparticles to entangled cis-1,4-polyisoprene and polydimethylsiloxane on the overall rheology of nanocomposites are discussed. In addition, insights about the relaxation of the host polymer chains and transport properties of nanoparticles in entangled polymer nanocomposites are presented. The nanoparticles are found to act as effective plasticizers for their entangled linear hosts, and below a critical, chemistry and molecular-weight dependent particle volume fraction, lead to reduced viscosity, glass transition temperature, number of entanglements, and polymer relaxation time. We also find that the particle motions in the polymer host are hyperdiffusive and at the nanoparticle length scale, the polymer host acts like a simple, ideal fluid and the composites\\' viscosity rises with increasing particle concentration. © 2012 The Royal Society of Chemistry.

  4. Laboratory Exercise for Studying the Morphology of Heat-Denatured and Amyloid Aggregates of Lysozyme by Atomic Force Microscopy

    Science.gov (United States)

    Gokalp, Sumeyra; Horton, William; Jónsdóttir-Lewis, Elfa B.; Foster, Michelle; Török, Marianna

    2018-01-01

    To facilitate learning advanced instrumental techniques, essential tools for visualizing biomaterials, a simple and versatile laboratory exercise demonstrating the use of Atomic Force Microscopy (AFM) in biomedical applications was developed. In this experiment, the morphology of heat-denatured and amyloid-type aggregates formed from a low-cost…

  5. Evaluation of several techniques to modify denatured muscle tissue to obtain a scaffold for peripheral nerve regeneration

    NARCIS (Netherlands)

    Meek, MF; den Dunnen, WFA; Schakenraad, JM; Robinson, PH

    The aim of this study was to (1) evaluate the effect of several preparation techniques of denatured muscle tissue to obtain an open three-dimensional structure, and (2) test if this scaffold is suitable for peripheral nerve regeneration. Four samples (A-D) of muscle tissue specimens were evaluated

  6. Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods

    Directory of Open Access Journals (Sweden)

    Yan-Song Gao

    2010-06-01

    Full Text Available The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK thermal denaturation were studied by differential scanning calorimetry (DSC, CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK. The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

  7. Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

    NARCIS (Netherlands)

    Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Stieger, Markus; Janssen, Anja E.M.

    2016-01-01

    The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60,

  8. The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

    Science.gov (United States)

    Fischer, T H

    1983-01-01

    To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy. PMID:6309139

  9. Clay-enhanced DNA separation in low-molecular-weight poly(N,N-dimethylacrylamide) solution by capillary electrophoresis.

    Science.gov (United States)

    Liang, D; Song, L; Chen, Z; Chu, B

    2001-06-01

    The effect of the separation medium in capillary electrophoresis consisting of a low-molecular-mass poly(N,N-dimethylacrylamide) (PDMA) solution on the DNA separation by adding a small amount of montmorillonite clay into the polymer matrix is presented. On the separation of the pBR322/HaeIII digest, both the resolution and the efficiency were increased by adding 2.5-5.0 x 10(-5) g/mL clay into the 5% w/v PDMA with a molecular mass of only 100 K. Moreover, there was no increase in the migration time of DNA fragments. Similar results were observed by using a C-terminated pGEM-3Zf(+) sequencing DNA sample in a sequencing buffer. Experimental data also showed that the addition of clay increased the viscosity of the polymer solution. We attribute this effect to the structural change of the polymer matrix caused by the exfoliated clay sheets, whereby the thin clay sheets function like a "dynamic cross-linking plate" for the PDMA chains and effectively increase the apparent molecular mass of PDMA.

  10. 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

  11. 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

  12. Polymer translocation under a pulling force: Scaling arguments and threshold forces

    Science.gov (United States)

    Menais, Timothée

    2018-02-01

    DNA translocation through nanopores is one of the most promising strategies for next-generation sequencing technologies. Most experimental and numerical works have focused on polymer translocation biased by electrophoresis, where a pulling force acts on the polymer within the nanopore. An alternative strategy, however, is emerging, which uses optical or magnetic tweezers. In this case, the pulling force is exerted directly at one end of the polymer, which strongly modifies the translocation process. In this paper, we report numerical simulations of both linear and structured (mimicking DNA) polymer models, simple enough to allow for a statistical treatment of the pore structure effects on the translocation time probability distributions. Based on extremely extended computer simulation data, we (i) propose scaling arguments for an extension of the predicted translocation times τ ˜N2F-1 over the moderate forces range and (ii) analyze the effect of pore size and polymer structuration on translocation times τ .

  13. A novel technique using DNA denaturation to detect multiply induced single-strand breaks in a hydrated plasmid DNA molecule by X-ray and 4He2+ ion irradiation

    International Nuclear Information System (INIS)

    Yokoya, A.; Shikazono, N.; Fujii, K.; Noguchi, M.; Urushibara, A.

    2011-01-01

    To detect multiple single-strand breaks (SSBs) produced in plasmid DNA molecules by direct energy deposition from radiation tracks, we have developed a novel technique using DNA denaturation by which irradiated DNA is analysed as single-strand DNA (SS-DNA). The multiple SSBs that arise in both strands of DNA, but do not induce a double-strand break, are quantified as loss of SS-DNA using agarose gel electrophoresis. We have applied this method to X-ray and 4 He 2+ ion-irradiated samples of fully hydrated pUC18 plasmid DNA. The fractions of both SS-DNA and closed circular DNA (CC-DNA) exponentially decrease with the increasing dose of X rays and 4 He 2+ ions. The efficiency of the loss of SS-DNA was half that of CC-DNA for both types of irradiation, indicating that one of two strands in DNA is not broken when one SSB is produced in CC-DNA by irradiation. Contrary to our initial expectation, these results indicate that SSBs are not multiply induced even by high linear energy transfer radiation distributed in both strands. (authors)

  14. Blood grouping based on PCR methods and agarose gel electrophoresis.

    Science.gov (United States)

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  15. Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

    International Nuclear Information System (INIS)

    Su Rongguo; Lin Jinming; Qu Feng; Chen Zhifeng; Gao Yunhua; Yamada, Masaaki

    2004-01-01

    In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis

  16. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  17. Microchip Electrophoresis at Elevated Temperatures and High Separation Field Strengths

    Science.gov (United States)

    Mitra, Indranil; Marczak, Steven P.; Jacobson, Stephen C.

    2014-01-01

    We report free-solution microchip electrophoresis performed at elevated temperatures and high separation field strengths. We used microfluidic devices with 11-cm long separation channels to conduct separations at temperatures between 22 (ambient) and 45 °C and field strengths from 100 to 1000 V/cm. To evaluate separation performance, N-glycans were used as a model system and labeled with 8-aminopyrene-1,3,6-trisulfonic acid to impart charge for electrophoresis and render them fluorescent. Typically, increased diffusivity at higher temperatures leads to increased axial dispersion and poor separation performance; however, we demonstrate that sufficiently high separation field strengths can be used to offset the impact of increased diffusivity in order to maintain separation efficiency. Efficiencies for these free-solution separations are the same at temperatures of 25, 35, and 45 °C with separation field strengths ≥500 V/cm. PMID:24114979

  18. Versatile electrophoresis-based self-test platform.

    Science.gov (United States)

    Guijt, Rosanne M

    2015-03-01

    Lab on a Chip technology offers the possibility to extract chemical information from a complex sample in a simple, automated way without the need for a laboratory setting. In the health care sector, this chemical information could be used as a diagnostic tool for example to inform dosing. In this issue, the research underpinning a family of electrophoresis-based point-of-care devices for self-testing of ionic analytes in various sample matrices is described [Electrophoresis 2015, 36, 712-721.]. Hardware, software, and methodological chances made to improve the overall analytical performance in terms of accuracy, precision, detection limit, and reliability are discussed. In addition to the main focus of lithium monitoring, new applications including the use of the platform for veterinary purposes, sodium, and for creatinine measurements are included. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. An improved interface for capillary zone electrophoresis-mass spectrometry

    International Nuclear Information System (INIS)

    Smith, R.D.; Loo, J.A.; Barinaga, C.J.; Udseth, H.R.

    1988-06-01

    We have recently developed an improved electrospray ionization interface for capillary electrophoresis mass-spectrometry (CZE-MS). Our initial interface employed a vacuum deposited metal film at the exit of the capillary to make an electrical contact with he eluting buffer and establish the electrospray field gradient. This interface did, however, impose significant limitations on the range of capillary electrophoretic (CE) separations that could be performed. To circumvent these limitations, an interface that does not require a metalized tip was designed nd developed. In the new approach, the electrical contact at the column exit is made through a flowing liquid sheath. The principal advantage of this interface is that it allows operation with a much broader range of electrophoresis conditions. The sheath flow can be readily varied in both composition and volume. An electrospray ionization spectrum is given for a previously intractable buffer solution. 5 refs., 2 figs

  20. 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.

  1. A simple gel electrophoresis method for separating polyhedral gold nanoparticles

    Science.gov (United States)

    Kim, Suhee; Lee, Hye Jin

    2015-07-01

    In this paper, a simple approach to separate differently shaped and sized polyhedral gold nanoparticles (NPs) within colloidal solutions via gel electrophoresis is described. Gel running parameters for separating efficiently gold NPs including gel composition, added surfactant types and applied voltage were investigated. The plasmonic properties and physical structure of the separated NPs extracted from the gel matrix were then investigated using transmission electron microscopy (TEM) and UV-vis spectrophotometry respectively. Data analysis revealed that gel electrophoresis conditions of a 1.5 % agarose gel with 0.1 % sodium dodecyl sulfate (SDS) surfactant under an applied voltage of 100 V resulted in the selective isolation of ~ 50 nm polyhedral shaped gold nanoparticles. Further efforts are underway to apply the method to purify biomolecule-conjugated polyhedral Au NPs that can be readily used for NP-enhanced biosensing platforms.

  2. 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.

  3. Sheathless interface for coupling capillary electrophoresis with mass spectrometry

    Science.gov (United States)

    Wang, Chenchen; Tang, Keqi; Smith, Richard D.

    2014-06-17

    A sheathless interface for coupling capillary electrophoresis (CE) with mass spectrometry is disclosed. The sheathless interface includes a separation capillary for performing CE separation and an emitter capillary for electrospray ionization. A portion of the emitter capillary is porous or, alternatively, is coated to form an electrically conductive surface. A section of the emitter capillary is disposed within the separation capillary, forming a joint. A metal tube, containing a conductive liquid, encloses the joint.

  4. 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.

  5. Electrophoresis today and tomorrow: helping biologists’ dreams come true

    Czech Academy of Sciences Publication Activity Database

    Klepárník, Karel; Boček, Petr

    2010-01-01

    Roč. 32, č. 3 (2010), s. 218-226 ISSN 0265-9247 R&D Projects: GA ČR GA203/08/1536; GA AV ČR IAA400310609; GA AV ČR IAA400310703; GA AV ČR KAN400310651 Institutional research plan: CEZ:AV0Z40310501 Keywords : capillary electrophoresis * isoelectric focusing * isotachophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.479, year: 2010

  6. Electrohydrodynamics and other hydrodynamic phenomena in continuous-flow electrophoresis

    International Nuclear Information System (INIS)

    Saville, D.A.

    1982-01-01

    The process known as continuous flow electrophoresis employs an electric field to separate the constituents of particulate samples suspended in a liquid. Complications arise because the electric field generates temperature gradients due to Joule heating and derives an electrohydrodynamic crossflow. Several aspects of the flow are discussed including entrance effects, hydrodynamic stability and a flow restructuring due to the combined effects of buoyancy and the crossflow. 10 references

  7. Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

    OpenAIRE

    Swerdlow, H; Gesteland, R

    1990-01-01

    Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequenci...

  8. Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

    OpenAIRE

    Brumley, R L; Smith, L M

    1991-01-01

    A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The appl...

  9. Automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis.

    Science.gov (United States)

    Sahore, Vishal; Sonker, Mukul; Nielsen, Anna V; Knob, Radim; Kumar, Suresh; Woolley, Adam T

    2018-01-01

    We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (μCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a μCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined μCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and μCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. Graphical abstract Pressure-actuated integrated microfluidic devices have been developed for automated solid-phase extraction, fluorescent labeling, and microchip electrophoresis of preterm birth biomarkers.

  10. 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.

  11. Phylogenetic reconstruction of South American felids defined by protein electrophoresis.

    Science.gov (United States)

    Slattery, J P; Johnson, W E; Goldman, D; O'Brien, S J

    1994-09-01

    Phylogenetic associations among six closely related South American felid species were defined by changes in protein-encoding gene loci. We analyzed proteins isolated from skin fibroblasts using two-dimensional electrophoresis and allozymes extracted from blood cells. Genotypes were determined for multiple individuals of ocelot, margay, tigrina, Geoffroy's cat, kodkod, and pampas cat at 548 loci resolved by two-dimensional electrophoresis and 44 allozyme loci. Phenograms were constructed using the methods of Fitch-Margoliash and neighbor-joining on a matrix of Nei's unbiased genetic distances for all pairs of species. Results of a relative-rate test indicate changes in two-dimensional electrophoresis data are constant among all South American felids with respect to a hyena outgroup. Allelic frequencies were transformed to discrete character states for maximum parsimony analysis. Phylogenetic reconstruction indicates a major split occurred approximately 5-6 million years ago, leading to three groups within the ocelot lineage. The earliest divergence led to Leopardus tigrina, followed by a split between an ancestor of an unresolved trichotomy of three species (Oncifelis guigna, O. geoffroyi, and Lynchailuris colocolo) and a recent common ancestor of Leopardus pardalis and L. wiedii. The results suggest that modern South American felids are monophyletic and evolved rapidly after the formation of the Panama land bridge between North and South America.

  12. 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

  13. 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.

  14. An analytical model for enantioseparation process in capillary electrophoresis

    Science.gov (United States)

    Ranzuglia, G. A.; Manzi, S. J.; Gomez, M. R.; Belardinelli, R. E.; Pereyra, V. D.

    2017-12-01

    An analytical model to explain the mobilities of enantiomer binary mixture in capillary electrophoresis experiment is proposed. The model consists in a set of kinetic equations describing the evolution of the populations of molecules involved in the enantioseparation process in capillary electrophoresis (CE) is proposed. These equations take into account the asymmetric driven migration of enantiomer molecules, chiral selector and the temporary diastomeric complexes, which are the products of the reversible reaction between the enantiomers and the chiral selector. The solution of these equations gives the spatial and temporal distribution of each species in the capillary, reproducing a typical signal of the electropherogram. The mobility, μ, of each specie is obtained by the position of the maximum (main peak) of their respective distributions. Thereby, the apparent electrophoretic mobility difference, Δμ, as a function of chiral selector concentration, [ C ] , can be measured. The behaviour of Δμ versus [ C ] is compared with the phenomenological model introduced by Wren and Rowe in J. Chromatography 1992, 603, 235. To test the analytical model, a capillary electrophoresis experiment for the enantiomeric separation of the (±)-chlorpheniramine β-cyclodextrin (β-CD) system is used. These data, as well as, other obtained from literature are in closed agreement with those obtained by the model. All these results are also corroborate by kinetic Monte Carlo simulation.

  15. Detecting irradiation of seeds using microgel electrophoresis (a collaborative trial)

    International Nuclear Information System (INIS)

    Cerda, H.; Haine, H.E.; Jones, J.L.

    1995-06-01

    Preservation of certain foods by irradiation is permitted in the United Kingdom. However, all irradiated foods must be labelled as such, to ensure consumer choice. To help enforce labelling, a variety of methods have been developed for distinguishing between irradiated and non-irradiated foods. In preliminary trials, microgel electrophoresis -a simple method of assessing DNA damage - has shown considerable promise in this respect. This report describes microgel electrophoresis, and details results obtained in a blind trial carried out in collaboration with the Swedish University of Agricultural Sciences. Microgel electrophoresis facilitates analysis of the leakage of DNA from cells extracted from food material. In irradiated samples, the DNA is fragmented and will leak from cells in an electric current. This leakage can be seen as a 'comet' when the stained gel is viewed with a microscope. The size and shape of the comet can be used to estimate the irradiation dose administered to the sample. In non-irradiated samples the DNA is less fragmented, will tend not to leak from the cells and will not form a comet. (author)

  16. Automated Lab-on-a-Chip Electrophoresis System

    Science.gov (United States)

    Willis, Peter A.; Mora, Maria; Greer, Harold F.; Fisher, Anita M.; Bryant, Sherrisse

    2012-01-01

    Capillary electrophoresis is an analytical technique that can be used to detect and quantify extremely small amounts of various biological molecules. In the search for biochemical traces of life on other planets, part of this search involves an examination of amino acids, which are the building blocks of life on Earth. The most sensitive method for detecting amino acids is the use of laser induced fluorescence. However, since amino acids do not, in general, fluoresce, they first must be reacted with a fluorescent dye label prior to analysis. After this process is completed, the liquid sample then must be transported into the electrophoresis system. If the system is to be reused multiple times, samples must be added and removed each time. In typical laboratories, this process is performed manually by skilled human operators using standard laboratory equipment. This level of human intervention is not possible if this technology is to be implemented on extraterrestrial targets. Microchip capillary electrophoresis (CE) combined with laser induced fluorescence detection (LIF) was selected as an extremely sensitive method to detect amino acids and other compounds that can be tagged with a fluorescent dye. It is highly desirable to package this technology into an integrated, autonomous, in situ instrument capable of performing CE-LIF on the surface of an extraterrestrial body. However, to be fully autonomous, the CE device must be able to perform a large number of sample preparation and analysis operations without the direct intervention of a human.

  17. Comparison of lipoprotein electrophoresis and apolipoprotein e genotyping in investigating dysbetalipoproteinemia

    International Nuclear Information System (INIS)

    Ahmed, F.; Kadiki, A.E.

    2017-01-01

    Dysbetalipoproteinemia is often associated with apolipoprotein E2E2 homozygosity; however, lipoprotein electrophoresis may also be used to assist in the diagnosis. The aim of this study was to compare apolipoprotein E (apo E) genotyping and lipoprotein electrophoresis in investigating dysbetalipoproteinemia. Data were collected over a three-year period from a lipid clinic in a tertiary referral centre and reviewed for apo E genotyping and lipoprotein electrophoresis. Sixty-two patients had both apo E genotyping and lipoprotein electrophoresis. Of these, 16 patients showed broad beta band on electrophoresis. However, only 3 of them had apo E2E2 homozygosity on genotyping. Lipoprotein electrophoresis and apo E genotyping results showed poor concordance. This was primarily due to visual interpretation error of lipoprotein electrophoresis which may over diagnose dysbetalipoproteinemia. (author)

  18. Comparison of Lipoprotein Electrophoresis and Apolipoprotein E Genotyping in Investigating Dysbetalipoproteinemia.

    Science.gov (United States)

    Ahmed, Farhan; El-Kadiki, Alia; Gibbons, Stephen

    2017-06-01

    Dysbetalipoproteinemia is often associated with apolipoprotein E2E2 homozygosity; however, lipoprotein electrophoresis may also be used to assist in the diagnosis. The aim of this study was to compare apolipoprotein E (apo E) genotyping and lipoprotein electrophoresis in investigating dysbetalipoproteinemia. Data were collected over a three-year period from a lipid clinic in a tertiary referral centre and reviewed for apo E genotyping and lipoprotein electrophoresis. Sixty-two patients had both apo E genotyping and lipoprotein electrophoresis. Of these, 16 patients showed broad beta band on electrophoresis. However, only 3 of them had apo E2E2 homozygosity on genotyping. Lipoprotein electrophoresis and apo E genotyping results showed poor concordance. This was primarily due to visual interpretation error of lipoprotein electrophoresis which may over diagnose dysbetalipoproteinemia.

  19. Applications of space-electrophoresis in medicine. [for cellular separations in molecular biology

    Science.gov (United States)

    Bier, M.

    1976-01-01

    The nature of electrophoresis is reviewed and potential advances realizable in the field of biology and medicine from a space electrophoresis facility are examined. The ground-based applications of electrophoresis: (1) characterization of an ionized species; (2) determination of the quantitative composition of a complex mixture; and (3) isolation of the components of a mixture, separation achieved on the basis of the difference in transport rates is reviewed. The electrophoresis of living cells is considered, touching upon the following areas: the separation of T and B lymphocytes; the genetic influence on mouse lymphocyte mobilities; the abnormal production of specific and monoclonal immunoproteins; and the study of cancer. Schematic diagrams are presented of three types of electrophoresis apparatus: the column assembly for the static electrophoresis experiment on the Apollo-Soyuz mission, the continuous flow apparatus used in the same mission and a miniaturized electrophoresis apparatus.

  20. Electrohydrodynamic properties of succinoglycan as probed by fluorescence correlation spectroscopy, potentiometric titration and capillary electrophoresis.

    Science.gov (United States)

    Duval, Jérôme F L; Slaveykova, Vera I; Hosse, Monika; Buffle, Jacques; Wilkinson, Kevin J

    2006-10-01

    The electrostatic, hydrodynamic and conformational properties of aqueous solutions of succinoglycan have been analyzed by fluorescence correlation spectroscopy (FCS), proton titration, and capillary electrophoresis (CE) over a large range of pH values and electrolyte (NaCl) concentrations. Using the theoretical formalism developed previously for the electrokinetic properties of soft, permeable particles, a quantitative analysis for the electro-hydrodynamics of succinoglycan is performed by taking into account, in a self-consistent manner, the measured values of the diffusion coefficients, electric charge densities, and electrophoretic mobilities. For that purpose, two limiting conformations for the polysaccharide in solution are tested, i.e. succinoglycan behaves as (i) a spherical, random coil polymer or (ii) a rodlike particle with charged lateral chains. The results show that satisfactory modeling of the titration data for ionic strengths larger than 50 mM can be accomplished using both geometries over the entire range of pH values. Electrophoretic mobilities measured for sufficiently large pH values (pH > 5-6) are in line with predictions based on either model. The best manner to discriminate between these two conceptual models is briefly discussed. For low pH values (pH < 5), both models indicate aggregation, resulting in an increase of the hydrodynamic permeability and a decrease of the diffusion coefficient.

  1. Optimization of capillary zone electrophoresis for charge heterogeneity testing of biopharmaceuticals using enhanced method development principles.

    Science.gov (United States)

    Moritz, Bernd; Locatelli, Valentina; Niess, Michele; Bathke, Andrea; Kiessig, Steffen; Entler, Barbara; Finkler, Christof; Wegele, Harald; Stracke, Jan

    2017-12-01

    CZE is a well-established technique for charge heterogeneity testing of biopharmaceuticals. It is based on the differences between the ratios of net charge and hydrodynamic radius. In an extensive intercompany study, it was recently shown that CZE is very robust and can be easily implemented in labs that did not perform it before. However, individual characteristics of some examined proteins resulted in suboptimal resolution. Therefore, enhanced method development principles were applied here to investigate possibilities for further method optimization. For this purpose, a high number of different method parameters was evaluated with the aim to improve CZE separation. For the relevant parameters, design of experiments (DoE) models were generated and optimized in several ways for different sets of responses like resolution, peak width and number of peaks. In spite of product specific DoE optimization it was found that the resulting combination of optimized parameters did result in significant improvement of separation for 13 out of 16 different antibodies and other molecule formats. These results clearly demonstrate generic applicability of the optimized CZE method. Adaptation to individual molecular properties may sometimes still be required in order to achieve optimal separation but the set screws discussed in this study [mainly pH, identity of the polymer additive (HPC versus HPMC) and the concentrations of additives like acetonitrile, butanolamine and TETA] are expected to significantly reduce the effort for specific optimization. 2017 The Authors. Electrophoresis published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Electrophoresis- and FRET-Based Measures of Serpin Polymerization.

    Science.gov (United States)

    Faull, Sarah V; Brown, Anwen E; Haq, Imran; Irving, James A

    2017-01-01

    Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

  3. Structural and dynamical study about denatured states of yeast phosphoglycerate kinase by neutrons scattering and X-rays; Etude structurale et dynamique des etats denatures de la phosphoglycerate kinase de levure par diffusion des neutrons et des rayons X

    Energy Technology Data Exchange (ETDEWEB)

    Receveur, V

    1997-04-28

    During a long time, the neutron scattering and X-rays techniques have not been used for the studies bearing on the folding of proteins. The compactness and the globularness of a protein are two structural characteristics describing the denatured states and the intermediate states of folding, and the neutrons and x-rays scattering are probably the two techniques the most appropriate to give this kind of information; they are sensible to the spatial extent and to the molecules compactness, and to their general shape. For these three or four last years, the works using these techniques are increasing, giving precious knowledge on the different steps of folding and on the interactions stabilizing the denatured or intermediate states. This thesis falls into this category. (N.C.).

  4. Microgel polymer composite fibres

    OpenAIRE

    Kehren, Dominic

    2014-01-01

    In this thesis some novel ideas and advancements in the field of polymer composite fibres, specifically microgel-based polymer composite fibres have been achieved. The main task was to investigate and understand the electrospinning process of microgels and polymers and the interplay of parameter influences, in order to fabricate reproducible and continuously homogenous composite fibres. The main aim was to fabricate a composite material which combines the special properties of polymer fibres ...

  5. Pu Denaturing by Transmutation of MA in FBR Multi-cycle

    Energy Technology Data Exchange (ETDEWEB)

    Meiliza, Yoshitalia; Saito, Masaki; Sagara, Hiroshi [Tokyo Institute of Technology, 2-12-1-N1-1 Ookayama, Meguro-ku, Tokyo, 1528550 (Japan)

    2009-06-15

    Pu accumulation and its recycling is important in the term of energy resources, however one of the most sensitive issues is non-proliferation in the future fuel cycle based on fast breeder reactor (FBR). The present paper utilizes Protected Pu Production (P{sup 3}) concept for the production of {sup 238}Pu and {sup 242}Pu by Minor Actinides (MA) transmutation to enhance the proliferation resistance of Pu in the fuel. Increase in the {sup 238}Pu and {sup 242}Pu isotopic fraction creates a high rate of internal heat generation by alpha decay (DH) and/or a high neutron source of spontaneous fission (SFN) in Pu that would be encountered during manufacturing and maintaining of nuclear explosive device. The feasibility of denaturing of Pu by MA transmutation in medium size FBR has been studied from the viewpoint of even-mass number Pu accumulation during multi-cycle of Pu and MA. The proliferation resistance property of Pu is also evaluated based on the specific decay heat and spontaneous fission neutron, compared with the reference criteria. In present paper, the P{sup 3} technology based on multi-recycled Pu and MA is compared with the conventional technology based on multi-recycled Pu only. The detail of mass balance behavior is, however, beyond the scope of the present paper. (authors)

  6. Precise determination of protein extinction coefficients under native and denaturing conditions using SV-AUC.

    Science.gov (United States)

    Hoffmann, Andreas; Grassl, Kerstin; Gommert, Janine; Schlesak, Christian; Bepperling, Alexander

    2018-04-17

    The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coefficient for a given protein are either based on the theoretical prediction utilizing the amino acid sequence or the photometric determination combined with a measurement of absolute protein concentration. Here, we report on an improved SV-AUC based method utilizing an analytical ultracentrifuge equipped with absorbance and Rayleigh interference optics. Global fitting of datasets helped to overcome some of the obstacles encountered with the traditional method employing synthetic boundary cells. Careful calculation of dn/dc values taking glycosylation and solvent composition into account allowed the determination of the extinction coefficients of monoclonal antibodies and an Fc-fusion protein under native as well as under denaturing conditions. An intra-assay precision of 0.9% and an accuracy of 1.8% compared to the theoretical value was achieved for monoclonal antibodies. Due to the large number of data points of a single dataset, no meaningful difference between the ProteomeLab XL-I and the new Optima AUC platform could be observed. Thus, the AUC-based approach offers a precise, convenient and versatile alternative to conventional methods like total amino acid analysis (AAA).

  7. Detection of urea-induced internal denaturation of dsDNA using solid-state nanopores.

    Science.gov (United States)

    Singer, Alon; Kuhn, Heiko; Frank-Kamenetskii, Maxim; Meller, Amit

    2010-11-17

    The ability to detect and measure dsDNA thermal fluctuations is of immense importance in understanding the underlying mechanisms responsible for transcription and replication regulation. We describe here the ability of solid-state nanopores to detect sub-nanometer changes in DNA structure as a result of chemically enhanced thermal fluctuations. In this study, we investigate the subtle changes in the mean effective diameter of a dsDNA molecule with 3-5 nm solid-state nanopores as a function of urea concentration and the DNA's AT content. Our studies reveal an increase in the mean effective diameter of a DNA molecule of approximately 0.6 nm at 8.7 M urea. In agreement with the mechanism of DNA local denaturation, we observe a sigmoid dependence of these effects on urea concentration. We find that the translocation times in urea are markedly slower than would be expected if the dynamics were governed primarily by viscous effects. Furthermore, we find that the sensitivity of the nanopore is sufficient to statistically differentiate between DNA molecules of nearly identical lengths differing only in sequence and AT content when placed in 3.5 M urea. Our results demonstrate that nanopores can detect subtle structural changes and are thus a valuable tool for detecting differences in biomolecules' environment.

  8. Detection of urea-induced internal denaturation of dsDNA using solid-state nanopores

    International Nuclear Information System (INIS)

    Singer, Alon; Kuhn, Heiko; Frank-Kamenetskii, Maxim; Meller, Amit

    2010-01-01

    The ability to detect and measure dsDNA thermal fluctuations is of immense importance in understanding the underlying mechanisms responsible for transcription and replication regulation. We describe here the ability of solid-state nanopores to detect sub-nanometer changes in DNA structure as a result of chemically enhanced thermal fluctuations. In this study, we investigate the subtle changes in the mean effective diameter of a dsDNA molecule with 3-5 nm solid-state nanopores as a function of urea concentration and the DNA's AT content. Our studies reveal an increase in the mean effective diameter of a DNA molecule of approximately 0.6 nm at 8.7 M urea. In agreement with the mechanism of DNA local denaturation, we observe a sigmoid dependence of these effects on urea concentration. We find that the translocation times in urea are markedly slower than would be expected if the dynamics were governed primarily by viscous effects. Furthermore, we find that the sensitivity of the nanopore is sufficient to statistically differentiate between DNA molecules of nearly identical lengths differing only in sequence and AT content when placed in 3.5 M urea. Our results demonstrate that nanopores can detect subtle structural changes and are thus a valuable tool for detecting differences in biomolecules' environment.

  9. CalFitter: a web server for analysis of protein thermal denaturation data.

    Science.gov (United States)

    Mazurenko, Stanislav; Stourac, Jan; Kunka, Antonin; Nedeljkovic, Sava; Bednar, David; Prokop, Zbynek; Damborsky, Jiri

    2018-05-14

    Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a limited number of relevant experimental tools. Widely-used experimental techniques, such as calorimetry or spectroscopy, critically depend on a proper data analysis. Currently, there are only separate data analysis tools available for each type of experiment with a limited model selection. To address this problem, we have developed the CalFitter web server to be a unified platform for comprehensive data fitting and analysis of protein thermal denaturation data. The server allows simultaneous global data fitting using any combination of input data types and offers 12 protein unfolding pathway models for selection, including irreversible transitions often missing from other tools. The data fitting produces optimal parameter values, their confidence intervals, and statistical information to define unfolding pathways. The server provides an interactive and easy-to-use interface that allows users to directly analyse input datasets and simulate modelled output based on the model parameters. CalFitter web server is available free at https://loschmidt.chemi.muni.cz/calfitter/.

  10. Formation of Polyphenol-Denatured Protein Flocs in Alcohol Beverages Sweetened with Refined Cane Sugars.

    Science.gov (United States)

    Eggleston, Gillian; Triplett, Alexa

    2017-11-08

    The sporadic appearance of floc from refined, white cane sugars in alcohol beverages remains a technical problem for both beverage manufacturers and sugar refiners. Cane invert sugars mixed with 60% pure alcohol and water increased light scattering by up to ∼1000-fold. Insoluble and soluble starch, fat, inorganic ash, oligosaccharides, Brix, and pH were not involved in the prevailing floc-formation mechanism. Strong polynomial correlations existed between the haze floc and indicator values (IVs) (color at 420 nm pH 9.0/color at pH 4.0-an indirect measure of polyphenolic and flavonoid colorants) (R 2 = 0.815) and protein (R 2 = 0.819) content of the invert sugars. Ethanol-induced denaturation of the protein exposed hydrophobic polyphenol-binding sites that were further exposed when heated to 80 °C. A tentative mechanism for floc formation was advanced by molecular probing with a haze (floc) active protein and polyphenol as well as polar, nonpolar, and ionic solvents.

  11. Modulation of the Extent of Cooperative Structural Change During Protein Folding by Chemical Denaturant.

    Science.gov (United States)

    Jethva, Prashant N; Udgaonkar, Jayant B

    2017-09-07

    Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N ↔ I N ↔ I 2 ↔ U model, in which structural changes occur noncooperatively during the N ↔ I N and I N ↔ I 2 transitions, and occur cooperatively during the I 2 ↔ U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.

  12. Effect of free cysteine on the denaturation and aggregation of holo α-lactalbumin

    DEFF Research Database (Denmark)

    Nielsen, Line R.; Lund, Marianne N.; Davies, Michael J.

    2018-01-01

    α-Lactalbumin (α-LA) is a key commercial whey protein for nutritional purposes. The holo protein (calcium saturated) is considered the most heat stable whey protein, capable of refolding from unfolded states under many conditions. This is due to the absence of free thiols (cysteine residues......) that are typically involved in thermal aggregation and thiol–disulphide exchange reactions of other whey proteins. Heating (0–120 min at 90 °C, pH 7.0) holo α-LA generates free thiols through thermal cleavage of disulphide bonds, resulting in aggregates comprising unfolded α-LA species. The addition of free cysteine...... promotes the formation of soluble aggregates, effectively decreasing the holding time required to reach a particular aggregate size in a dose-dependent manner (0.35–1.4 mM cysteine). Excess cysteine (≥14 mM) causes a destabilisation of α-LA, shown by decreased denaturation temperature and gel formation...

  13. Size distribution of DNA molecules recovered from non-denaturing filter elution

    International Nuclear Information System (INIS)

    Bloecher, D.; Iliakis, G.

    1991-01-01

    DNA fragments removed from the filter during non-denaturing filter elution were collected and loaded on top of neutral sucrose gradients. Their size distribution was determined by low-speed centrifugation in neutral sucrose gradients. The average size of eluted DNA was found to be approximately 110 S; the average size of DNA collected after short elution times was found to be slightly larger than after long elution times. It is concluded that the size of eluted DNA fragments is not correlated with elution rate, and it is proposed that shear forces generated at the filter pores cause degradation of the DNA. Comparison of sedimentation profiles of carefully prepared cellular DNA before and after elution revealed that generated shear forces during elution break down DNA to an extent equivalent to around 20 000 DNA double-strand breaks (dsb) per G 1 cell. The size of DNA fragments decreased with increasing radiation dose; five times more dsb were found than expected after exposure to radiation alone. It is proposed that excess of dsb may derive from the transformation of other radiation-induced lesions to dsb under the action of shear forces generated during elution. (author)

  14. Programmable self-assembly of carbon nanotubes assisted by reversible denaturation of a protein

    International Nuclear Information System (INIS)

    Nithiyasri, P; Parthasarathy, M; Balaji, K; Brindha, P

    2012-01-01

    Self-assembly of pristine multi-walled carbon nanotubes (CNTs) in aqueous dispersion using a protein, bovine serum albumin (BSA), has been demonstrated. Step-wise conformational changes in BSA as a function of temperature have been deployed to direct the assembly of nanotubes. More specifically, CNTs distributed randomly in native BSA at 35 °C as well as completely denatured BSA solution at 80 °C self-assemble in the intermediate temperature range of 45–65 °C, as evident from scanning and transmission electron microscopy. Fourier transform infrared (FTIR) and fluorescence studies indicate significant changes in the α-helical content of the protein with respect to the amide I and II bands and tryptophan emission intensity, respectively. The stability of CNT dispersion in BSA solution has been attributed to the hydrophobic interaction between nanotubes and the protein molecule by adding sodium cholate to the dispersion. Moreover, a mechanism based on electrostatic repulsion between BSA-bound CNTs has been proposed for the thermally reversible assembly of CNTs in BSA solution based on evidence from zeta potential measurements and FTIR spectroscopy. Thus the present report demonstrates bio-mimetic self-assembly of as-synthesized CNTs using changes in surface charge and conformation of an unfolding protein for biomedical applications and nanobiotechnology. (paper)

  15. Protein Denaturation on p-T Axes--Thermodynamics and Analysis.

    Science.gov (United States)

    Smeller, László

    2015-01-01

    Proteins are essential players in the vast majority of molecular level life processes. Since their structure is in most cases substantial for their correct function, study of their structural changes attracted great interest in the past decades. The three dimensional structure of proteins is influenced by several factors including temperature, pH, presence of chaotropic and cosmotropic agents, or presence of denaturants. Although pressure is an equally important thermodynamic parameter as temperature, pressure studies are considerably less frequent in the literature, probably due to the technical difficulties associated to the pressure studies. Although the first steps in the high-pressure protein study have been done 100 years ago with Bridgman's ground breaking work, the field was silent until the modern spectroscopic techniques allowed the characterization of the protein structural changes, while the protein was under pressure. Recently a number of proteins were studied under pressure, and complete pressure-temperature phase diagrams were determined for several of them. This review summarizes the thermodynamic background of the typical elliptic p-T phase diagram, its limitations and the possible reasons for deviations of the experimental diagrams from the theoretical one. Finally we show some examples of experimentally determined pressure-temperature phase diagrams.

  16. The quaternary structure of the recombinant bovine odorant-binding protein is modulated by chemical denaturants.

    Directory of Open Access Journals (Sweden)

    Olga V Stepanenko

    Full Text Available A large group of odorant-binding proteins (OBPs has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl. Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M. This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

  17. Thermosetting Phthalocyanine Polymers

    Science.gov (United States)

    Fohlen, G.; Parker, J.; Achar, B.

    1985-01-01

    Group of phthalocyanine polymers resist thermal degradation. Polymers expected semiconducting. Principal applications probably in molded or laminated parts that have to withstand high temperatures. Polymers made from either of two classes of monomer: Bisphthalonitriles with imide linkages or Bisphthalonitriles with ester-imide linkages.

  18. based gel polymer electrolytes

    Indian Academy of Sciences (India)

    (PVdF) as a host polymer, lithium perchlorate (LiClO4), lithium triflate ... TG/DTA studies showed the thermal stability of the polymer electrolytes. .... are observed while comparing pure XRD spectra with .... batteries as its operating temperature is normally in the .... chain ion movements and the conductivity of the polymer.

  19. Aerogel / Polymer Composite Materials

    Science.gov (United States)

    Williams, Martha K. (Inventor); Smith, Trent M. (Inventor); Fesmire, James E. (Inventor); Roberson, Luke B. (Inventor); Clayton, LaNetra M. (Inventor)

    2017-01-01

    The invention provides new composite materials containing aerogels blended with thermoplastic polymer materials at a weight ratio of aerogel to thermoplastic polymer of less than 20:100. The composite materials have improved thermal insulation ability. The composite materials also have better flexibility and less brittleness at low temperatures than the parent thermoplastic polymer materials.

  20. Structural and dynamical study about denatured states of yeast phosphoglycerate kinase by neutrons scattering and X-rays

    International Nuclear Information System (INIS)

    Receveur, V.

    1997-01-01

    During a long time, the neutron scattering and X-rays techniques have not been used for the studies bearing on the folding of proteins. The compactness and the globularness of a protein are two structural characteristics describing the denatured states and the intermediate states of folding, and the neutrons and x-rays scattering are probably the two techniques the most appropriate to give this kind of information; they are sensible to the spatial extent and to the molecules compactness, and to their general shape. For these three or four last years, the works using these techniques are increasing, giving precious knowledge on the different steps of folding and on the interactions stabilizing the denatured or intermediate states. This thesis falls into this category. (N.C.)

  1. Electroluminescence of Multicomponent Conjugated Polymers. 1. Roles of Polymer/Polymer Interfaces in Emission Enhancement and Voltage-Tunable Multicolor Emission in Semiconducting Polymer/Polymer Heterojunctions

    National Research Council Canada - National Science Library

    Zhang, Xuejun, Ph.D

    1999-01-01

    Effects of the electronic structure of polymer/polymer interfaces on the electroluminescence efficiency and tunable multicolor emission of polymer heterojunction light-emitting diodes were explored...

  2. Nanoporous polymer electrolyte

    Science.gov (United States)

    Elliott, Brian [Wheat Ridge, CO; Nguyen, Vinh [Wheat Ridge, CO

    2012-04-24

    A nanoporous polymer electrolyte and methods for making the polymer electrolyte are disclosed. The polymer electrolyte comprises a crosslinked self-assembly of a polymerizable salt surfactant, wherein the crosslinked self-assembly includes nanopores and wherein the crosslinked self-assembly has a conductivity of at least 1.0.times.10.sup.-6 S/cm at 25.degree. C. The method of making a polymer electrolyte comprises providing a polymerizable salt surfactant. The method further comprises crosslinking the polymerizable salt surfactant to form a nanoporous polymer electrolyte.

  3. Polymer friction Molecular Dynamics

    DEFF Research Database (Denmark)

    Sivebæk, Ion Marius; Samoilov, Vladimir N.; Persson, Bo N. J.

    We present molecular dynamics friction calculations for confined hydrocarbon solids with molecular lengths from 20 to 1400 carbon atoms. Two cases are considered: a) polymer sliding against a hard substrate, and b) polymer sliding on polymer. In the first setup the shear stresses are relatively...... independent of molecular length. For polymer sliding on polymer the friction is significantly larger, and dependent on the molecular chain length. In both cases, the shear stresses are proportional to the squeezing pressure and finite at zero load, indicating an adhesional contribution to the friction force....

  4. Biostable glucose permeable polymer

    DEFF Research Database (Denmark)

    2017-01-01

    A new biostable glucose permeable polymer has been developed which is useful, for example, in implantable glucose sensors. This biostable glucose permeable polymer has a number of advantageous characteristics and, for example, does not undergo hydrolytic cleavage and degradation, thereby providing...... a composition that facilitates long term sensor stability in vivo. The versatile characteristics of this polymer allow it to be used in a variety of contexts, for example to form the body of an implantable glucose sensor. The invention includes the polymer composition, sensor systems formed from this polymer...

  5. Protective role of microRNA-29a in denatured dermis and skin fibroblast cells after thermal injury

    Directory of Open Access Journals (Sweden)

    Jie Zhou

    2016-03-01

    Full Text Available Our previous study has suggested that downregulated microRNA (miR-29a in denatured dermis might be involved in burn wound healing. However, the exact role of miR-29a in healing of burn injury still remains unclear. Here, we found that expression of miR-29a was notably upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury, and thereafter gradually downregulated compared with control group. By contrast, the expression of collagen, type I, alpha 2 (COL1A2 and vascular endothelial growth factor (VEGF-A were first reduced and subsequently upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury. We further identified COL1A2 as a novel target of miR-29a, which is involved in type I collagen synthesis, and showed that miR-29a negatively regulated the expression level of COL1A2 in skin fibroblast cells. In addition, VEGF-A, another target gene of miR-29a, was also negatively mediated by miR-29a in skin fibroblast cells. Inhibition of miR-29a expression significantly promoted the proliferation and migration of skin fibroblast cells after thermal injury, and knockdown of COL1A2 and VEGF-A reversed the effects of miR-29a on the proliferation and migration of skin fibroblast cells. Furthermore, we found that Notch2/Jagged2 signaling was involved in miR-29a response to burn wound healing. Our findings suggest that downregulated miR-29a in denatured dermis may help burn wound healing in the later phase, probably via upregulation of COL1A2 and VEGF-A expression, which can further enhance type I collagen synthesis and angiogenesis.

  6. Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: A chemical chaperone at atomic resolution

    OpenAIRE

    Bennion, Brian J.; Daggett, Valerie

    2004-01-01

    Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea ...

  7. Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

    Energy Technology Data Exchange (ETDEWEB)

    Luna Z, D [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    2005-07-01

    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  8. Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

    Science.gov (United States)

    Inoue, H; Hirobe, M

    1987-05-29

    The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

  9. A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium Ions

    Science.gov (United States)

    Nielsen, Anders D.; Pusey, Marc L.; Fuglsang, Claus C.; Westh, Peter

    2003-01-01

    The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.

  10. Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life.

    Directory of Open Access Journals (Sweden)

    M Luisa Romero-Romero

    Full Text Available The relationship between the denaturation temperatures of proteins (Tm values and the living temperatures of their host organisms (environmental temperatures: TENV values is poorly understood. Since different proteins in the same organism may show widely different Tm's, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm's are oftentimes found to correlate with TENV's but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm's for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

  11. NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

    Science.gov (United States)

    Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

    2003-01-01

    The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

  12. Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

    Energy Technology Data Exchange (ETDEWEB)

    Luna Z, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)]. e-mail: dlz@nuclear.inin.mx

    2005-07-01

    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  13. Fluorination of polymers

    International Nuclear Information System (INIS)

    Du Toit, F.J.

    1991-01-01

    Polyethylene and polypropylene were reacted with elemental fluorine under carefully controlled conditions to produce fluorocarbon polymers. Fluorination of polymer films resulted in fluorination of only the outer surfaces of the films, while the reaction of elemental fluorine with powdered hydrocarbon polymers produced perfluorocarbon polymers. Existing and newly developed techniques were used to characterize the fluorinated polymers. It was shown that the degree of fluorination was influenced by the surface area of the hydrocarbon material, the concentration, of the fluorine gas, and the time and temperature of fluorination. A fluidized-bed reactor used for the fluorination of polymer powders effectively increased the reaction rate. The surface tension and the oxygen permeability of the fluorinated polymers were studied. The surface tension of hydrocarbon polymers was not influenced by different solvents, but the surface tension of fluorinated polymers was affected by the type of solvent that was used. There were indications that the surface tension was affected by oxygen introduced into the polymer surface during fluorination. Fluorination lowered the permeability of oxygen through hydrocarbon polymers. 55 refs., 51 figs., 26 tabs

  14. Advanced polymers in medicine

    CERN Document Server

    Puoci, Francesco

    2014-01-01

    The book provides an up-to-date overview of the diverse medical applications of advanced polymers. The book opens by presenting important background information on polymer chemistry and physicochemical characterization of polymers. This serves as essential scientific support for the subsequent chapters, each of which is devoted to the applications of polymers in a particular medical specialty. The coverage is broad, encompassing orthopedics, ophthalmology, tissue engineering, surgery, dentistry, oncology, drug delivery, nephrology, wound dressing and healing, and cardiology. The development of polymers that enhance the biocompatibility of blood-contacting medical devices and the incorporation of polymers within biosensors are also addressed. This book is an excellent guide to the recent advances in polymeric biomaterials and bridges the gap between the research literature and standard textbooks on the applications of polymers in medicine.

  15. Antiviral Polymer Therapeutics

    DEFF Research Database (Denmark)

    Smith, Anton Allen Abbotsford

    2014-01-01

    polymerized in a controlled manner with carrier monomers of historically proven biocompatible polymers. The carrier polymers, the loading of ribavirin as well as the size of the polymer were varied systematically with the aid of an automated synthesis platform. These polymers were tested in a cellular assay...... of reversible-addition-fragmentation chain transfer polymerization, which not only controls the size of polymer, but also allows the introduction of a terminal amine on the polymer which can be used for further conjugation. This has allowed for not only fluorescent labeling of the polymer, but also protein...... is mediated through specific transporters, it is thought that the accumulation can be alleviated through the attachment of ribavirin to a macromolecule. To this end, ribavirin was enzymatically modified into a monomer compatible with controlled polymerization techniques. The ribavirin monomers were...

  16. 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

  17. Improved protocol for isolation of Campylobacter spp. from retail broiler meat and use of pulsed field gel electrophoresis for the typing of isolates.

    Science.gov (United States)

    Oyarzabal, Omar A; Williams, Aretha; Zhou, Ping; Samadpour, Mansour

    2013-10-01

    To improve the detection of Campylobacter spp. in retail broiler meat, a reference method (R subsamples) based on the enrichment of 25 g of meat in Bolton broth at 42°C under microaerobiosis was compared with an alternative method (A subsamples) consisting in the rinsing of meat samples for 30s in buffered peptone water with antimicrobials with incubation at 42°C under aerobiosis. One piece of meat (breasts, tenderloins and thighs) was rinse in experiment 1 (A1) and two pieces in experiment 2 (A2). Campylobacter spp. were isolated on agar plates and identified by PCR. Retail samples in Alabama had less prevalence (P ≤ 0.05) than samples in the state of Washington. The percentage of positive was higher (P ≤ 0.05) in A than in R subsamples and rinsing two pieces of meat yielded the highest percentage of positive subsamples. R subsamples showed variations in the prevalence by product. However, A subsamples had similar prevalence of positives among products compare to the result from reference method. More Campylobacter coli isolates were collected in A2 subsamples. Pulse field gel electrophoresis (PFGE) was used as subtyping method to study the genome similarity among the isolates from all methods. A larger diversity of isolates were detected by PFGE in A2 subsamples. Denaturing gradient gel electrophoresis analysis suggested that the initial bacterial populations of the meat samples impact the final bacterial profile after enrichment. Rinsing broiler meats was less time consuming, required less sample preparation and was more sensitive than the reference method for the isolation of naturally occurring Campylobacter spp. This new method could help with epidemiological and intervention studies to control Campylobacter spp. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. The application of methylation specific electrophoresis (MSE to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

    Directory of Open Access Journals (Sweden)

    Yokoyama Seiya

    2012-02-01

    Full Text Available Abstract Background Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Methods Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE. The MSE method is comprised of the following steps: (a bisulfite treatment of genomic DNA, (b amplification of the target DNA by a nested PCR approach and (c applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. Result The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is Conclusions The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

  19. Polymer reaction engineering, an integrated approach

    NARCIS (Netherlands)

    Meyer, T.; Keurentjes, J.T.F.; Meyer, T.; Keurentjes, J.T.F.

    2005-01-01

    Summary This chapter contains sections titled: Polymer Materials A Short History of Polymer Reaction Engineering The Position of Polymer Reaction Engineering Toward Integrated Polymer Reaction Engineering The Disciplines in Polymer Reaction Engineering The Future: Product-inspired Polymer Reaction

  20. A method for easily customizable gradient gel electrophoresis.

    Science.gov (United States)

    Miller, Andrew J; Roman, Brandon; Norstrom, Eric

    2016-09-15

    Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for routine analysis without the need for specialized mixing equipment. The method allows for easily customizable gradients which can be optimized for specific polypeptide resolution requirements. Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and the time and resources saved with this method in place of traditional gradient mixing, or the purchase of pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Human muscle proteins: analysis by two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Giometti, C.S.; Danon, M.J.; Anderson, N.G.

    1983-09-01

    Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be cause by generalized muscle-fiber damage.

  2. Separation and determination of some carboxylic acids by capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Sladkov, V.; Fourest, B

    2006-07-01

    Separation and determination of some organic acids, mono-carboxylic (formic and acetic), dicarboxylic (oxalic and tartaric), tricarboxylic (citric) acids and aromatic acids (phtalic, benzoic, mellitic and trimellitic), by capillary electrophoresis are reviewed. The method development parameters, such as separation and injection mode, are discussed. Special attention is paid to the comparison of different detection types (spectroscopic and electrochemical). The optimisation of the carrier electrolyte composition (choice of carrier electrolyte, effect of pH, ionic strength, electro-osmotic flow modifier) is treated. Different additives (alkali-earth and transition metal ions, cyclodextrins and alcohol), which are often used for improving organic acid separation, are also considered. (authors)

  3. Multivalent weak electrolytes - risky background electrolytes for capillary zone electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Beckers, J. L.; Boček, Petr

    2002-01-01

    Roč. 23, č. 12 (2002), s. 1942-1946 ISSN 0173-0835 R&D Projects: GA ČR GA203/99/0044; GA ČR GA203/02/0023; GA ČR GA203/01/0401; GA AV ČR IAA4031703; GA AV ČR IAA4031103 Institutional research plan: CEZ:AV0Z4031919 Keywords : background electrolytes * capillary zone electrophoresis * multivalent electrolytes Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.325, year: 2002

  4. Separation and determination of some carboxylic acids by capillary electrophoresis

    International Nuclear Information System (INIS)

    Sladkov, V.; Fourest, B.

    2006-01-01

    Separation and determination of some organic acids, mono-carboxylic (formic and acetic), dicarboxylic (oxalic and tartaric), tricarboxylic (citric) acids and aromatic acids (phtalic, benzoic, mellitic and trimellitic), by capillary electrophoresis are reviewed. The method development parameters, such as separation and injection mode, are discussed. Special attention is paid to the comparison of different detection types (spectroscopic and electrochemical). The optimisation of the carrier electrolyte composition (choice of carrier electrolyte, effect of pH, ionic strength, electro-osmotic flow modifier) is treated. Different additives (alkali-earth and transition metal ions, cyclodextrins and alcohol), which are often used for improving organic acid separation, are also considered. (authors)

  5. Separation and quantification of cellulases and hemicellulases by capillary electrophoresis

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Kutter, Jörg Peter; Olsson, Lisbeth

    2003-01-01

    Cellulases and hemicellulases are two classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. Both classes of enzymes consist of a subset of classes of which the fungi produce several enzymes with varying molecular mass and pI but similar enzymatic activities....... Current methods are limited in their ability to quantify all of these enzymes when all are present simultaneously in a mixture. Five different cellulases (two cellobiohydrolases and three endoglucanases) and one hemicellulase (endoxylanase) were separated using capillary electrophoresis (CE) in a fused...

  6. Capillary electrophoresis-MALDI interface based on inkjet technology

    Science.gov (United States)

    Vannatta, Michael W.; Whitmore, Colin D.; Dovichi, Norman J.

    2010-01-01

    An ink jet printer valve and nozzle were used to deliver matrix and sample from an electrophoresis capillary onto a MALDI plate. The system was evaluated by separation of a set of standard peptides. That separation generated up to 40,000 theoretical plates in less than three minutes. Detection limits were 500 amol using an ABI TOF-TOF instrument and 2 fmol for an ABI Q-TOF instrument. Over 70% coverage was obtained for the tryptic digest of α-lactalbumin in less than 2.5 minutes. PMID:19960472

  7. Recent advances of capillary electrophoresis in pharmaceutical analysis.

    Science.gov (United States)

    Suntornsuk, Leena

    2010-09-01

    This review covers recent advances of capillary electrophoresis (CE) in pharmaceutical analysis. The principle, instrumentation, and conventional modes of CE are briefly discussed. Advances in the different CE techniques (non-aqueous CE, microemulsion electrokinetic chromatography, capillary isotachophoresis, capillary electrochromatography, and immunoaffinity CE), detection techniques (mass spectrometry, light-emitting diode, fluorescence, chemiluminescence, and contactless conductivity), on-line sample pretreatment (flow injection) and chiral separation are described. Applications of CE to assay of active pharmaceutical ingredients (APIs), drug impurity testing, chiral drug separation, and determination of APIs in biological fluids published from 2008 to 2009 are tabulated.

  8. Capillaries for use in a multiplexed capillary electrophoresis system

    Science.gov (United States)

    Yeung, E.S.; Chang, H.T.; Fung, E.N.

    1997-12-09

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  9. Electronic imaging systems for quantitative electrophoresis of DNA

    International Nuclear Information System (INIS)

    Sutherland, J.C.

    1989-01-01

    Gel electrophoresis is one of the most powerful and widely used methods for the separation of DNA. During the last decade, instruments have been developed that accurately quantitate in digital form the distribution of materials in a gel or on a blot prepared from a gel. In this paper, I review the various physical properties that can be used to quantitate the distribution of DNA on gels or blots and the instrumentation that has been developed to perform these tasks. The emphasis here is on DNA, but much of what is said also applies to RNA, proteins and other molecules. 36 refs

  10. A novel temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device

    DEFF Research Database (Denmark)

    Bu, Minqiang; R. Perch-Nielsen, Ivan; Sørensen, Karen Skotte

    steps to achieve a rapid ramping between the temperature steps for DNA denaturation, annealing and extension. The temperature dynamics within the microfluidic PCR chamber was characterized and the overshooting and undershooting parameters were optimized using the temperature dependent fluorescence......We present a new temperature control method capable of effectively shortening the thermal cycling time of polymerase chain reaction (PCR) in a disposable polymer microfluidic device with external heater and temperature sensor. The method employs optimized temperature overshooting and undershooting...

  11. A temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device

    DEFF Research Database (Denmark)

    Bu, Minqiang; Perch-Nielsen, Ivan R.; Sørensen, Karen Skotte

    2013-01-01

    steps to achieve a rapid ramping between the temperature steps for DNA denaturation, annealing and extension. The temperature dynamics within the microfluidic PCR chamber was characterized and the overshooting and undershooting parameters were optimized using the temperature-dependent fluorescence......We present a temperature control method capable of effectively shortening the thermal cycling time of polymerase chain reaction (PCR) in a disposable polymer microfluidic device with an external heater and a temperature sensor. The method employs optimized temperature overshooting and undershooting...

  12. Transition from uranium to denatured uranium/thorium fuel in an existing PWR

    International Nuclear Information System (INIS)

    Walters, M.A.

    1982-01-01

    The purpose of this research was to determine whether it is possible to make a gradual transition from uranium to denatured uranium/thorium (DUTH) fuel in an existing PWR by adding DUTH assemblies during each scheduled refueling and, if the transition is possible, to develop a general procedure for making it. The feasibility of the transition was established by identifying acceptable refueling schemes for a series of transition cores, and in the process, a method for identifying acceptable schemes evolved. The utility of the method was then demonstrated by applying it to a standard reactor operating under normal conditions. The vehicle used to examine proposed fuel mixtures and to select acceptable ones was a set of one-dimensional computer codes. The core was modeled as a set of five concentric fuel zones with a reflector. Fuel mixtures were proposed and the computer codes were used to determine whether a mixture was acceptable, i.e., whether it had the desired k-effective and flux and power distributions. The parameters allowed to vary in selection of proposed fuel mixtures were enrichment of fresh fuel assemblies, number of uranium and DUTH assemblies added during each refueling, and distribution of fuel in the core. Results of the research showed that a gradual transition is possible. Furthermore, there is a method that allows the identification of fuel mixtures that are likely to be acceptable. It requires the calculation of K-infinity for the entire proposed core and for some of its regions. These values of K-infinity and relationships developed in this research can be used to predict the flux distribution and the final k-effective for the proposed fuel mixture

  13. Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation

    International Nuclear Information System (INIS)

    Jackson, E J; Coussios, C-C; Cleveland, R O

    2014-01-01

    Thermal ablation by high intensity focused ultrasound (HIFU) has a great potential for the non-invasive treatment of solid tumours. Due to the high pressure amplitudes involved, nonlinear acoustic effects must be understood and the relevant medium property is the parameter of nonlinearity B/A. Here, B/A was measured in ex vivo bovine liver, over a heating/cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique, which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane wave through liver and B/A was chosen so that numerical simulations matched the measured waveforms. To create plane-wave conditions, sinusoidal bursts were transmitted by a 100 mm diameter 1.125 MHz unfocused transducer and measured using a 15 mm diameter 2.25 MHz broadband transducer in the near field. Attenuation and sound-speed were calculated using a reflected pulse from the smaller transducer using the larger transducer as the reflecting interface. Results showed that attenuation initially decreased with heating then increased after denaturation, the sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. The B/A data disagree with other reports that show a significant change and we suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity. (paper)

  14. A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.

    Science.gov (United States)

    Brewer, J M; Wampler, J E

    2001-03-14

    The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

  15. Star-Branched Polymers (Star Polymers)

    KAUST Repository

    Hirao, Akira; Hayashi, Mayumi; Ito, Shotaro; Goseki, Raita; Higashihara, Tomoya; Hadjichristidis, Nikolaos

    2015-01-01

    The synthesis of well-defined regular and asymmetric mixed arm (hereinafter miktoarm) star-branched polymers by the living anionic polymerization is reviewed in this chapter. In particular, much attention is being devoted to the synthetic

  16. Polymer dynamics from synthetic polymers to proteins

    Indian Academy of Sciences (India)

    Abstract. Starting from the standard model of polymer motion – the Rouse model – .... reptation and the escape processes (creep motion) from the tube. .... scattering curves from an arrangement of small mesoscopic spheres also allows a.

  17. Rapid monitoring of autolysis process of proteases by capillary electrophoresis.

    Science.gov (United States)

    Chen, Xiu-Lan; Shun, Cai-Yun; Zhang, Yu-Zhong; Gao, Pei-Ji

    2003-10-01

    A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 degrees C-50 degrees C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 degrees C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 degrees C. Similar results were obtained when MCP-01 was incubated at 20 degrees C, 40 degrees C and 50 degrees C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.

  18. Canine serum protein patterns using high-resolution electrophoresis (HRE).

    Science.gov (United States)

    Abate, O; Zanatta, R; Malisano, T; Dotta, U

    2000-03-01

    Serum protein values were determined in 26 healthy dogs using agarose gel electrophoresis (SPE), splitting the electrophoretic separation into six regions: albumin, alpha(1), alpha(2), beta(1), beta(2)and gamma globulins. High-resolution electrophoresis (HRE) was used to separate single proteins. Serum proteins from dogs (26 healthy and 20 affected by various diseases) were then characterized by electrophoretic immunofixation (IFE) and Sudan black staining on HRE film. Haemoglobin and normal canine plasma and serum were used to identify haptoglobin and fibrinogen, respectively. In the standard pattern, determined by HRE, the following proteins were identified: albumin, alpha(1)-lipoprotein (alpha(1)-region), haptoglobin and alpha(2)-macroglobulin (alpha(2)-region), beta -lipoprotein and C3 (beta(1)-region), transferrin and IgM (beta(2)-region), IgG (mostly in gamma -region and partly in beta(2)-region). The HRE pattern shown by healthy dogs could be compared with those of dogs affected by various diseases to obtain clinical information. Copyright 2000 Harcourt Publishers Ltd.

  19. Tilted hexagonal post arrays: DNA electrophoresis in anisotropic media.

    Science.gov (United States)

    Chen, Zhen; Dorfman, Kevin D

    2014-02-01

    Using Brownian dynamics simulations, we show that DNA electrophoresis in a hexagonal array of micron-sized posts changes qualitatively when the applied electric field vector is not coincident with the lattice vectors of the array. DNA electrophoresis in such "tilted" post arrays is superior to the standard "un-tilted" approach; while the time required to achieve a resolution of unity in a tilted post array is similar to an un-tilted array at a low-electric field strengths, this time (i) decreases exponentially with electric field strength in a tilted array and (ii) increases exponentially with electric field strength in an un-tilted array. Although the DNA dynamics in a post array are complicated, the electrophoretic mobility results indicate that the "free path," i.e. the average distance of ballistic trajectories of point-sized particles launched from random positions in the unit cell until they intersect the next post, is a useful proxy for the detailed DNA trajectories. The analysis of the free path reveals a fundamental connection between anisotropy of the medium and DNA transport therein that goes beyond simply improving the separation device. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Success and failure with phthalate buffers in capillary zone electrophoresis.

    Science.gov (United States)

    Bocek, P; Gebauer, P; Beckers, J L

    2001-04-01

    Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the migration of analytes is strongly deteriorated due to the presence of a system zone. System zones in phthalate buffers may be easily detected by UV detection and manifest themselves as peaks or dips. Peak shape diagrams are advantageously used for the prediction of the migration behavior of system zones in phthalate background electrolyte (BGE) systems at various pH. It is shown that the mobility of the system zone varies strongly with pH, is practically zero at pH values below 4 and above 7, and shows a maximum at pH 5. Thus, the system peak may coincide either with the peaks of various analytes or with the electroosmotic flow (EOF) peak. Experiments are given showing the effects of such coincidences as, e.g., zigzag detection patterns, double EOF peaks, and/or unusually broad peaks/dips. The message of this contribution is to show how to understand the electrophoretic properties of phthalate BGEs that, regardless of possible failure regions, may be successfully used in the analytical practice of capillary zone electrophoresis (CZE).