WorldWideScience

Sample records for standard agarose electrophoresis

  1. Nondenaturing agarose gel electrophoresis of RNA.

    Science.gov (United States)

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W

    2010-06-01

    INTRODUCTION Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. There are two common types of gel: polyacrylamide and agarose. For most applications involving RNAs of or =600 nucleotides, and are especially useful for analysis of mRNAs (e.g., by Northern blotting). RNA analysis on agarose gels is essentially identical to DNA analysis (except that the gel boxes used must be dedicated to RNA work or to other ribonuclease-free work). Here we describe the use of straightforward Tris borate, EDTA (TBE) gels for routine analysis. These gels are appropriate for determining the quantity and integrity of RNA before using it for other applications. This procedure should not be used to determine size with accuracy, because the RNA will not remain in its extended state throughout the run.

  2. 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

  3. Separation of long RNA by agarose-formaldehyde gel electrophoresis.

    Science.gov (United States)

    Mansour, Farrah H; Pestov, Dimitri G

    2013-10-01

    We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative "pK-matched" buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Preparative electrophoresis on linear polyacrylamide-agarose composite gels.

    Science.gov (United States)

    Shainoff, J R; Smejkal, G B; Mitkevich, O; DiBello, P M

    1996-01-01

    A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).

  5. A simple and effective SuperBuffer for DNA agarose electrophoresis.

    Science.gov (United States)

    Zhang, Jun-He; Wang, Fang; Wang, Tian-Yun

    2011-11-01

    In the paper, we describe a unique effective electrophoresis buffer for DNA agarose electrophoresis, called SuperBuffer. Using this buffer, electrophoresis could be performed within 10 min at voltages as high as 25V/cm. In addition, DNA fragments of different lengths could be isolated clearly even at lower agarose gel concentrations and the DNA recovery efficiency was higher than that of the TAE/TBE running buffers. The SuperBuffer still retained its electrophoretic effect even after several uses. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. 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.

  7. Electrophoresis of immunoglobulin G. Facilitated migration of minute amounts in agarose

    International Nuclear Information System (INIS)

    Weil, M.L.; Tyrrell, D.L.J.; Norrby, E.

    1978-01-01

    Migration of very small amounts of immunoglobulin (20 ng) is restricted in agarose electrophoresis. Incorporation of a stable protein matrix (rabbit gamma globulin 1 mg/ml) in the agarose permits unrestricted migration so that immunoelectrophoresis of this quantity of radiolabelled antibody is possible. Very small amounts of radiolabelled and non-radiolabelled antibody were subjected to successful crossed immunoelectrophoresis through barriers of antigen under conditions which provide favorable ratios of antibody to antigen. These methods should be useful for studies of antibody eluted from tissue in acquired and autoimmune diseases associated with tissue bound immunoglobulin. (Auth.)

  8. Analysis of Human Papillomavirus Genome Replication Using Two- and Three-Dimensional Agarose Gel Electrophoresis.

    Science.gov (United States)

    Henno, Liisi; Tombak, Eva-Maria; Geimanen, Jelizaveta; Orav, Marit; Ustav, Ene; Ustav, Mart

    2017-05-16

    This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two-dimensional and neutral/neutral/alkaline three-dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard-to-interpret signals of human papilloma virus replication intermediates obtained from two- and three-dimensional agarose gels. Examples of typical results and interpretation of replication intermediate patterns are included, and the outcomes of multiple-dimension assays are assessed using previously published experimental data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  9. Confirmation of soybean plastid rRNAs by formaldehyde denaturing agarose gel electrophoresis.

    Science.gov (United States)

    Zhu, Y Q; Zheng, Y; Chen, H B; Huang, L Q

    2014-10-27

    Owing to their prokaryotic origin, plastid rRNAs are mainly 23s/16s/5s rRNAs. We present a novel plant RNA isolation method in this paper. Also, not only the eukaryotic 28s (26s, 25s)/18s rRNAs but the prokaryotic 26s/23s rRNAs as well were demonstrated in a single sample for the first time by formaldehyde denaturing agarose gel electrophoresis.

  10. Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca

    2009-01-01

    A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses.Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 microg. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 microg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications.

  11. Simple and practical staining of DNA with GelRed in agarose gel electrophoresis.

    Science.gov (United States)

    Huang, Qing; Baum, Larry; Fu, Wei-Ling

    2010-01-01

    Although SYBR Gold or SYBR Green I have been used in the loading buffer as a DNA stain safer than ethidium bromide for agarose gel electrophoresis, electrophoretic mobility of DNA is altered and thus DNA fragment size cannot be accurately determined. A method using GelRed in the loading buffer was developed to stain DNA fragments in agarose gel electrophoresis. Among various concentrations of GelRed, SYBR Gold, or SYBR Green I tested in the loading buffer, only the highest tested concentration of GelRed, i.e., 100x GelRed, did not change band mobility. Evaluations using various sizes of PCR products at different concentrations further confirmed that 100x GelRed could be used to accurately determine DNA fragment size. The reagent can be stored at 4 degrees C for at least 1 year without a decrease in staining sensitivity. The 100x GelRed is a sensitive and safe alternative to ethidium bromide and better than either SYBR Gold or SYBR Green I for size determination in agarose gel electrophoresis. Our laboratory now uses the GelRed method routinely with great consistency and success.

  12. Response surface methodology-based optimisation of agarose gel electrophoresis for screening and electropherotyping of rotavirus.

    Science.gov (United States)

    Mishra, Vikas; Nag, Vijaya Lakshmi; Tandon, Ritu; Awasthi, Shally

    2010-04-01

    Management of rotavirus diarrhoea cases and prevention of nosocomial infection require rapid diagnostic method at the patient care level. Diagnostic tests currently available are not routinely used due to economic or sensitivity/specificity constraints. Agarose-based sieving media and running conditions were modulated by using central composite design and response surface methodology for screening and electropherotyping of rotaviruses. The electrophoretic resolution of rotavirus genome was calculated from input parameters characterising the gel matrix structure and running conditions. Resolution of rotavirus genome was calculated by densitometric analysis of the gel. The parameters at critical values were able to resolve 11 segmented rotavirus genome. Better resolution and electropherotypic variation in 11 segmented double-stranded RNA genome of rotavirus was detected at 1.96% (w/v) agarose concentration, 0.073 mol l(-1) ionic strength of Tris base-boric acid-ethylenediamine tetraacetic acid buffer (1.4x) and 4.31 h of electrophoresis at 4.6 V cm(-1) electric field strength. Modified agarose gel electrophoresis can replace other methods as a simplified alternative for routine detection of rotavirus where it is not in practice.

  13. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    Science.gov (United States)

    Vetcher, Alexandre A.; Srinivasan, Srimeenakshi; Vetcher, Ivan A.; Abramov, Semen M.; Kozlov, Mikhail; Baughman, Ray H.; Levene, Stephen D.

    2006-08-01

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

  14. Performance comparison of capillary and agarose gel electrophoresis for the identification and characterization of monoclonal immunoglobulins.

    Science.gov (United States)

    McCudden, Christopher R; Mathews, Stephanie P; Hainsworth, Shirley A; Chapman, John F; Hammett-Stabler, Catherine A; Willis, Monte S; Grenache, David G

    2008-03-01

    The objective of this study was to compare gel- and capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins (M proteins). Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis (AGE), capillary electrophoresis (CE), immunofixation electrophoresis (IFE), and subtraction immunotyping (IT). As a screening test for detecting M proteins, AGE and CE displayed similar sensitivity (91% and 92%, respectively). CE was less specific (74%) than AGE (81%). An analysis of interinterpreter agreement revealed that interpretations were more consistent using gel-based methods than capillary-based methods, with 80% of the gel interpretations being in complete (5/5) agreement compared with 67% of the capillary interpretations. After implementing the capillary-based methods, the number of tests per reportable result increased (from 1.58 to 1.73). CE is an analytically suitable alternative to AGE, but laboratories implementing it will need to continue IFE testing to characterize all M proteins detected by CE.

  15. Analysis of Replicating Mitochondrial DNA by In Organello Labeling and Two-Dimensional Agarose Gel Electrophoresis.

    Science.gov (United States)

    Holt, Ian J; Kazak, Lawrence; Reyes, Aurelio; Wood, Stuart R

    2016-01-01

    Our understanding of the mechanisms of DNA replication in a broad range of organisms and viruses has benefited from the application of two-dimensional agarose gel electrophoresis (2D-AGE). The method resolves DNA molecules on the basis of size and shape and is technically straightforward. 2D-AGE sparked controversy in the field of mitochondria when it revealed replicating molecules with lengthy tracts of RNA, a phenomenon never before reported in nature. More recently, radioisotope labeling of the DNA in the mitochondria has been coupled with 2D-AGE. In its first application, this procedure helped to delineate the "bootlace mechanism of mitochondrial DNA replication," in which processed mitochondrial transcripts are hybridized to the lagging strand template at the replication fork as the leading DNA strand is synthesized. This chapter provides details of the method, how it has been applied to date and concludes with some potential future applications of the technique.

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

    Science.gov (United States)

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

    2000-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Jania Bartosz

    2016-12-01

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

  18. Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis.

    Science.gov (United States)

    Halfmann, Randal; Lindquist, Susan

    2008-07-16

    Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms. Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity. However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE). This technique is finding widespread use for the detection and characterization of amyloid conformational variants. Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins. The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated. Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

  19. Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate.

    Science.gov (United States)

    Serwer, Philip; Wright, Elena T

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. High resolution melt analysis (HRMA; a viable alternative to agarose gel electrophoresis for mouse genotyping.

    Directory of Open Access Journals (Sweden)

    Nicole Thomsen

    Full Text Available Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.

  1. DNA agarose gel electrophoresis for antioxidant analysis: Development of a quantitative approach for phenolic extracts.

    Science.gov (United States)

    Silva, Sara; Costa, Eduardo M; Vicente, Sandra; Veiga, Mariana; Calhau, Conceição; Morais, Rui M; Pintado, Manuela E

    2017-10-15

    Most of the fast in vitro assays proposed to determine the antioxidant capacity of a compound/extract lack either biological context or employ complex protocols. Therefore, the present work proposes the improvement of an agarose gel DNA electrophoresis in order to allow for a quantitative estimation of the antioxidant capacity of pure phenolic compounds as well as of a phenolic rich extract, while also considering their possible pro-oxidant effects. The result obtained demonstrated that the proposed method allowed for the evaluation of the protection of DNA oxidation [in the presence of hydrogen peroxide (H 2 O 2 ) and an H 2 O 2 /iron (III) chloride (FeCl 3 ) systems] as well as for the observation of pro-oxidant activities, with the measurements registering interclass correlation coefficients above 0.9. Moreover, this method allowed for the characterization of the antioxidant capacity of a blueberry extract while demonstrating that it had no perceived pro-oxidant effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins.

    Science.gov (United States)

    Ramsey, Kathryn A; Rushton, Zachary L; Ehre, Camille

    2016-06-14

    Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

  3. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis.

    Science.gov (United States)

    Sanderson, Brian A; Araki, Naoko; Lilley, Jennifer L; Guerrero, Gilberto; Lewis, L Kevin

    2014-06-01

    Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (∼10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20-25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10-100 bp): high voltages and short run times produced sharper bands and higher resolution. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Affinophoresis in two-dimensional agarose gel electrophoresis: specific separation of biomolecules by a moving affinity ligand.

    Science.gov (United States)

    Shimura, K; Kasai, K

    1987-02-15

    Affinophoresis is an electrophoretic separation technique for biomolecules which uses an affinophore. An affinophore is a macromolecular polyelectrolyte bearing affinity ligands. It migrates rapidly in an electric field, and consequently the electrophoretic mobility of molecules having affinity for the ligand is specifically changed. This technique has now been incorporated in two-dimensional agarose gel electrophoresis in a procedure which utilizes normal electrophoresis in the first dimension and affinophoresis in the second dimension. Proteins which do not have affinity for the ligand migrate to locations along a diagonal line passing through the origin, whereas proteins which have affinity are carried away from the line by the affinophore. Accordingly, molecules having affinity for the ligand can be readily assigned. Trypsins contained in Pronase and pancreatin were separated by this procedure using an affinophore bearing a competitive inhibitor for trypsin, benzamidine, on a polyanionic molecule (a polyacrylic acid derivative).

  5. Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

    OpenAIRE

    Halfmann, Randal; Lindquist, Susan

    2008-01-01

    Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms. Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity. However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel El...

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

    Science.gov (United States)

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

    2007-03-01

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

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

    Science.gov (United States)

    Greaser, Marion L; Warren, Chad M

    2015-01-01

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

  8. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    Science.gov (United States)

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  9. Heparins, low-molecular-weight heparins, and other glycosaminoglycans analyzed by agarose gel electrophoresis and azure A-silver staining.

    Science.gov (United States)

    Wang, L; Malsch, R; Harenberg, J

    1997-01-01

    A sensitive, nonradioactive azure A-silver staining method combining agarose gel electrophoresis was established and evaluated. Unfractionated heparins (UFHs), low-molecular-weight heparins (LMWHs), heparan sulfate (HS), chondroitin sulfate A (CSA), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronic acid (HA) were analyzed. The detection limit of the method was 0.5 ng for heparin, LMWH, HA, CSA, and DS, 2 ng for KS, and 6 ng for HA in the 2-microliter sample volume. Dilution curves demonstrated linear correlation between the logarithm of the concentration of glycosaminoglycans (GAGs) and their optical absorbance at 548 nm. The linear ranges were 1 to 500 ng/microliter for heparins, LMWHs, HS, DS, and CSA, 3 to 500 ng/microliter for KS, and 8 to 500 ng/microliter for HA. GAGs have their characteristic migration patterns and their Rf value decreased from CSA to KS, DS, HS, heparin, and HA. The differences were described for heparins and LMWHs. LMWHs migrated faster and displayed broader bands than unfractionated heparins. It was also observed that some unfractionated heparins contained low sulfated GAGs as contamination, which seemed to be DS as judged by their migration patterns.

  10. Agarose gel electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA system as a new prognostic tool for periprosthetic osteolysisin revision arthroplasty

    Science.gov (United States)

    Chiva, A

    2013-01-01

    Rationale. Prevention of wear-mediated osteolysis, the most common complication in total joint arthroplasty, is a great challenge for orthopedic surgery. Despite the diversity of current biomarkers of periprosthetic osteolysis (products of wear, bone turnover and inflammatory biomarkers), the major interferences and the great amount of sample necessary for analysis limit their use in clinical practice. Objective. The aim of this paper is to present three new electrophoretic methods using Hyrys-Hydrasys SEBIA system that have been used for the first time in Electrophoresis Laboratory of our hospital in the analysis of joint fluid for the prevention of periprosthetic osteolysis in revision arthroplasty. Methods and results. Analytical aspects of agarose gel electrophoresis of joint fluid proteins and lipoproteins as well as SDS-agarose gel electrophoresis of joint fluid proteins, their performances and clinical value are presented. The decreased level of albumin and increased level of alpha1 and alpha2 globulins were frequent changes detected on SEBIA electropherograms and good indicator for the presence of an inflammatory reaction generated by particle debris. In addition, a slightly increase of LDL mobility could provide good information about a high oxidative stress. Moreover, the Ig G assessed by using SDS-agarose gel electrophoresis could be a potential biomarker for an immunological reaction towards orthopedic implants. Discussion. Electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA France system is a new analytical technique able to remove the most of current biomarkers disadvantages due to the determination of particular proteins (acute phase proteins, albumin, lipoproteins, and immunoglobulins) by using minimal amounts of joint fluid with minor interferences, minimal cost and rapid results. Abbreviations CTX, crosslinked C-telopeptide; IL- interleukins; Ig G, immunoglobulin G; LDL, low density lipoprotein; NTX, crosslinked N-telopeptide; PICP

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

    Science.gov (United States)

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

    2006-01-01

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

  12. 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.

  13. Standardizing electrophoresis conditions: how to eliminate a major source of error in the comet assay.

    Directory of Open Access Journals (Sweden)

    Gunnar Brunborg

    2015-06-01

    Full Text Available In the alkaline comet assay, cells are embedded in agarose, lysed, and then subjected to further processing including electrophoresis at high pH (>13. We observed very large variations of mean comet tail lengths of cell samples from the same population when spread on a glass or plastic substrate and subjected to electrophoresis. These variations might be cancelled out if comets are scored randomly over a large surface, or if all the comets are scored. The mean tail length may then be representative of the population, although its standard error is large. However, the scoring process often involves selection of 50 – 100 comets in areas selected in an unsystematic way from a large gel on a glass slide. When using our 96-sample minigel format (1, neighbouring sample variations are easily detected. We have used this system to study the cause of the comet assay variations during electrophoresis and we have defined experimental conditions which reduce the variations to a minimum. We studied the importance of various physical parameters during electrophoresis: (i voltage; (ii duration of electrophoresis; (iii electric current; (iv temperature; and (v agarose concentration. We observed that the voltage (V/cm varied substantially during electrophoresis, even within a few millimetres of distance between gel samples. Not unexpectedly, both the potential ( V/cm and the time were linearly related to the mean comet tail, whereas the current was not. By measuring the local voltage with microelectrodes a few millimetres apart, we observed substantial local variations in V/cm, and they increased with time. This explains the large variations in neighbouring sample comet tails of 25% or more. By introducing simple technology (circulation of the solution during electrophoresis, and temperature control, these variations in mean comet tail were largely abolished, as were the V/cm variations. Circulation was shown to be particularly important and optimal conditions

  14. Comparison of the capillary and agarose electrophoresis based multiple locus VNTR (variable number of tandem repeats) analysis (MLVA) on Mycobacterium bovis isolates.

    Science.gov (United States)

    Jenkins, A O; Venter, E H; Hutamo, K; Godfroid, J

    2010-09-28

    Electrophoretic techniques that can be used for genotyping of bacterial pathogens ranges from manual, low-cost, agarose gels to high-throughput capillary electrophoresis sequencing machines. These two methods are currently employed in the electrophoresis of PCR products used in multiple locus VNTR (variable number of tandem repeats) analysis (MLVA), i.e. the agarose electrophoresis (AE) and the capillary electrophoresis (CE). Some authors have suggested that clusters generated by AE are less reliable than those generated by CE and that the latter is a more sensitive technique than the former when typing Mycobacterium tuberculosis complex (MTC) isolates. Because such a claim could have significant consequences for investigators in this field, a comparison was made on 19 Belgian Mycobacterium bovis strains which had previously been genotyped using CE VNTR analysis. The VNTR profiles of the CE VNTR analysis were compared with those obtained by AE VNTR analysis at 14 VNTR loci. Our results indicated that there were no differences in copy numbers at all loci tested when the copy numbers obtained by the AE VNTR analysis were compared with those obtained by CE VNTR analysis. The use of AE VNTR analysis in mycobacterial genotyping does not alter the sensitivity of the MLVA technique compared with the CE VNTR analysis. The AE VNTR can therefore be regarded as a viable alternative in moderately equipped laboratories that cannot afford the expensive equipment required for CE VNTR analysis and data obtained by AE VNTR analysis can be shared between laboratories which use the CE VNTR method. (c) 2010 Elsevier B.V. All rights reserved.

  15. Purification and cloning of DNA fragments fractionated on agarose gels.

    Science.gov (United States)

    Griffin, H G; Gasson, M J

    1995-04-01

    Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.

  16. Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

    Science.gov (United States)

    Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

    2015-07-01

    Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

  17. Improvement of electrophoresis performance by spectral analysis ...

    African Journals Online (AJOL)

    This paper describes a new design of standard agarose gel electrophoresis procedure for nucleic acids analysis. The electrophoresis was improved by using the real-time spectral analysis of the samples to increase its performance. A laser beam illuminated the analysed sample at wavelength with the highest absorption of ...

  18. Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB:DNA complex studied by agarose gel electrophoresis.

    Science.gov (United States)

    Villagomez, Rodrigo; Hatti-Kaul, Rajni; Sterner, Olov; Almanza, Giovanna; Linares-Pastén, Javier A

    2015-01-01

    The nuclear factor κB (NF-κB) is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB - DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB-DNA complex. The most active compounds are substituted at C-3 (α-carbonyl), in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

  19. Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB:DNA complex studied by agarose gel electrophoresis.

    Directory of Open Access Journals (Sweden)

    Rodrigo Villagomez

    Full Text Available The nuclear factor κB (NF-κB is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB - DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB-DNA complex. The most active compounds are substituted at C-3 (α-carbonyl, in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

  20. Agarose gel purification of PCR products for denaturing gradient gel electrophoresis results in GC-clamp deletion.

    Science.gov (United States)

    Sun, Guowei; Xiao, Jinzhou; Lu, Man; Wang, Hongming; Chen, Xiaobing; Yu, Yongxin; Pan, Yingjie; Wang, Yongjie

    2015-01-01

    The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)-clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35 ± 0.19 °C) was less than that of PPFS (83.81 ± 0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples.

  1. Application of urea-agarose gel electrophoresis to select non-redundant 16S rRNAs for taxonomic studies: palladium(II) removal bacteria.

    Science.gov (United States)

    Assunção, Ana; Costa, Maria Clara; Carlier, Jorge Dias

    2016-03-01

    The 16S ribosomal RNA (rRNA) gene has been the most commonly used sequence to characterize bacterial communities. The classical approach to obtain gene sequences to study bacterial diversity implies cloning amplicons, selecting clones, and Sanger sequencing cloned fragments. A more recent approach is direct sequencing of millions of genes using massive parallel technologies, allowing a large-scale biodiversity analysis of many samples simultaneously. However, currently, this technique is still expensive when applied to few samples; therefore, the classical approach is still used. Recently, we found a community able to remove 50 mg/L Pd(II). In this work, aiming to identify the bacteria potentially involved in Pd(II) removal, the separation of urea/heat-denatured DNA fragments by urea-agarose gel electrophoresis was applied for the first time to select 16S rRNA-cloned amplicons for taxonomic studies. The major raise in the percentage of bacteria belonging to genus Clostridium sensu stricto from undetected to 21 and 41 %, respectively, for cultures without, with 5 and 50 mg/L Pd(II) accompanying Pd(II) removal point to this taxa as a potential key agent for the bio-recovery of this metal. Despite sulfate-reducing bacteria were not detected, the hypothesis of Pd(II) removal by activity of these bacteria cannot be ruled out because a slight decrease of sulfate concentration of the medium was verified and the formation of PbS precipitates seems to occur. This work also contributes with knowledge about suitable partial 16S rRNA gene regions for taxonomic studies and shows that unidirectional sequencing is enough when Sanger sequencing cloned 16S rRNA genes for taxonomic studies to genus level.

  2. Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.

    Science.gov (United States)

    Rio, Donald C

    2015-03-02

    In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA. © 2015 Cold Spring Harbor Laboratory Press.

  3. Low melting point agarose beads as a standard method for plantlet regeneration from protoplasts within the Cichorium genus.

    Science.gov (United States)

    Deryckere, Dieter; Eeckhaut, Tom; Van Huylenbroeck, Johan; Van Bockstaele, Erik

    2012-12-01

    A standard method has been developed with which we are able to fully regenerate protoplasts of different Cichorium species. For the first time, endive protoplasts have been regenerated into plantlets. Protoplast regeneration is essential for somatic hybridizations. In this study, a standard method for plantlet regeneration from Cichorium protoplasts was developed. We evaluated the effect of the low melting point agarose (LMPA) bead technique on the regeneration capacity of protoplasts of seven C. intybus and four C. endivia genotypes. The LMPA bead technique was more efficient than culture in liquid or solid medium and allowed us to obtain plating efficiencies up to 4.9 % in C. intybus genotypes and efficiencies of up to 0.7 % in C. endivia genotypes. Moreover, the LMPA bead technique offers great advantages over liquid and solid culture systems: the media can be readily refreshed, protoplasts can be monitored separately, and microcalli can easily be removed from the beads. This increased efficiency was observed for all of the 11 Cichorium genotypes tested. Shoot formation was induced more efficiently when using 0.5 mg l(-1) indole-3-acetic acid-enriched medium (up to 87.5 % of the protoplast-derived calli started shoot development) compared to 1-naphthaleneacetic acid-enriched medium. The LMPA bead technique optimized in this study enabled for the first time the full plantlet regeneration from protoplasts of C. endivia genotypes and increased the protoplast regenerating ability in other Cichorium species. This fine-tuned LMPA bead technique can therefore be applied for protoplast regeneration after protoplast fusions of the genus Cichorium.

  4. 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.

  5. 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.

  6. Ethidium Bromide Modifies The Agarose Electrophoretic Mobility of CAG•CTG Alternative DNA Structures Generated by PCR

    Directory of Open Access Journals (Sweden)

    Mário Gomes-Pereira

    2017-05-01

    Full Text Available The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.

  7. 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.

  8. Agarose Gel Size Selection for DNA Sequencing Libraries.

    Science.gov (United States)

    Mardis, Elaine; McCombie, W Richard

    2017-08-01

    Agarose gel electrophoresis may be used to purify fragmented genomic DNA after ligation of adaptors. After electrophoresis, the region of the gel containing the desired size range of DNA is excised, and the DNA is subsequently extracted from the gel and purified by passage through a spin column. © 2017 Cold Spring Harbor Laboratory Press.

  9. Bleach gel: a simple agarose gel for analyzing RNA quality.

    Science.gov (United States)

    Aranda, Patrick S; LaJoie, Dollie M; Jorcyk, Cheryl L

    2012-01-01

    RNA-based applications requiring high-quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the 'bleach gel' is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Recovery of DNA from agarose gel by trap method | Xia | African ...

    African Journals Online (AJOL)

    Recovery of DNA from agarose gel electrophoresis is a basic operation during molecular cloning. Circular or linear DNA fragments which vary from 1.5 to 6.5 kb and correspond to 1 kb marker can be recovered from 0.8 to 1.0% agarose gel smoothly with a simple and rapid trap method. The recovery efficiency could be ...

  11. Use of a commercial agarose gel for analysis of urinary glycosaminoglycans in mucopolysaccharidoses

    Directory of Open Access Journals (Sweden)

    Ana Carolina Breier

    Full Text Available ABSTRACT Mucopolysaccharidoses (MPS are a group of inherited metabolic disorders caused by deficiency of enzymes that degrade glycosaminoglycans (GAGs. Urinary excretion of GAGs is a common feature of MPS, and is considered their major biomarker. We aimed to adapt the GAG electrophoresis method to a commercial agarose gel which would be able to separate urinary GAGs in a simpler way with good sensitivity and reproducibility. Urine samples from patients previously diagnosed with MPS I, IV, and VI were used as electrophoretic standards. Samples from patients on enzyme replacement therapy (ERT were also assessed. Commercial agarose gel electrophoresis was effective, showing proper definition and separation of GAG bands. Detection sensitivity exceeded 0.1 µg and band reproducibility were consistent. GAG bands quantified in urine samples from patients on ERT correlated very strongly (correlation coefficient = 0.98 with total GAG concentrations. This application of gel electrophoresis demonstrates the possibility of monitoring patients with MPS treated with ERT by analyzing separately the GAGs excreted in urine. We suggest this process should be applied to MPS screening as well as to follow-up of patients on treatment.

  12. 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.

  13. Development and validation of an internationally-standardized, high-resolution capillary gel-based electrophoresis PCR-ribotyping protocol for Clostridium difficile.

    Directory of Open Access Journals (Sweden)

    Warren N Fawley

    Full Text Available PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping, so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies. Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.

  14. Hydrogen peroxide agarose gels for electrophoretic analysis of RNA.

    Science.gov (United States)

    Pandey, Renu; Saluja, Daman

    2017-10-01

    Efficient electrophoretic separation of isolated total RNA utilizes chemicals and agents to aid in nuclease free environment. However cost, extensive pre-run processing protocols as well as toxic byproducts limit the usage of such protocols. Moreover, these treatments affect the overall electrophoretic results by altering the conductivity of the running buffer and weaken the gel strength. We here provide a protocol for RNA visualization that obviates these shortcomings by preparation of agarose gel with hydrogen peroxide using the regular TAE buffer. The simple, inexpensive protocol exhibits superior results in a horizontal agarose gel electrophoresis. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Quantification in capillary electrophoresis-mass spectrometry : Long- and short-term variance components and their compensation using internal standards

    NARCIS (Netherlands)

    Ohnesorge, Jens; Sänger-van de Griend, Cari; Wätzig, Hermann

    Different approaches were chosen to examine ionization reproducibility of analytes after separation by capillary electrophoresis-mass spectrometry (CE-MS) in a commercially available sheath-flow electrospray interface. For this task three different standard samples were examined. Sample 1 contained

  16. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.

    Science.gov (United States)

    Imbeaud, Sandrine; Graudens, Esther; Boulanger, Virginie; Barlet, Xavier; Zaborski, Patrick; Eveno, Eric; Mueller, Odilo; Schroeder, Andreas; Auffray, Charles

    2005-03-30

    While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data.

  17. Caracterización de la diversidad genética en naranja y comparación del polimorfismo de microsatélites amplificados al azar (RAMs usando electroforesis de poliacrilamida y azarosa Characterization of the genetic diversity in orange, and comparison of polymorphism in randomly-amplifed microsatellites (RAMs, using polyacrylamide and agarose electrophoresis

    Directory of Open Access Journals (Sweden)

    Ana Cruz Morillo Coronado

    2009-10-01

    Full Text Available Se compararon las eficiencias de tres métodos de electroforesis en agarosa y poliacrilamida, usando la cámara pequeña de DNA Sequencing System y cámara grande OWL Sequi-Gen Sequencing Cell, en la detección del polimorfismo en 21 accesiones de naranja (Citrus sinensis con empleo del cebador CGA. El gel de poliacrilamida dio mejor resolución de los productos amplificados vía PCR producidos por RAMs. Este permitió una mejor detección de bandas de ADN polimórficas, lo que facilitó la identificación de la variabilidad genética. La electroforesis en agarosa puede ser más conveniente en otras aplicaciones, debido al bajo costo y fácil aplicación. El estudio de diversidad genética en naranja usando microsatélites RAMs diferenció 51 accesiones en siete grupos con 0.75 de similaridad y 0.25 de heterocigosidad, lo que revela bajo polimorfismo genético. La técnica RAMs permitió agrupar las accesiones en Comunes o Blancas, Navel y Pigmentadas o Sanguinas.We compared the efficiency of three methods of agarose and polyacrylamide electrophoresis (using the small tank of the DNA Sequencing System and the large OWL Sequi-Gen Sequencing Cell, for the detection of polymorphism in 21 accessions of orange (Citrus sinensis, using the primer CGA. The polyacrylamide gel gave better resolution of the PCR-amplified RAM products. This method allowed better detection of polymorphic DNA bands, facilitating the identification of genetic variability. The agarose electrophoresis may be more convenient in other applications, due to its low cost and easy implementation. The study of genetic diversity in orange using RAMs separated 51 accessions into seven groups with 0.75 similarity, and 0.25 heterozygosity, revealing low genetic polymorphism. The RAMs technique grouped the accessions into “Common or White”, “Navel” and “Pigmented or “Sanguine”.

  18. Composition of agarose substrate affects behavioral output of Drosophila larvae

    Directory of Open Access Journals (Sweden)

    Anthi Aristomenis Apostolopoulou

    2014-01-01

    Full Text Available In the last decade the Drosophila larva has evolved into a simple model organism offering the opportunity to integrate molecular genetics with systems neuroscience. This led to a detailed understanding of the functional neuronal networks for a number of sensory functions and behaviors including olfaction, vision, gustation and learning and memory. Typically, behavioral assays in use exploit simple Petri dish setups with either agarose or agar as a substrate. However, neither the quality nor the concentration of the substrate is generally standardized across these experiments and there is no data available on how larval behavior is affected by such different substrates. Here, we have investigated the effects of different agarose concentrations on several larval behaviors. We demonstrate that agarose concentration is an important parameter, which affects all behaviors tested: preference, feeding, learning and locomotion. Larvae can discriminate between different agarose concentrations, they feed differently on them, they can learn to associate an agarose concentration with an odor stimulus and crawl faster on a substrate of higher agarose concentration. Additionally, we have investigated the effect of agarose concentration on three quinine based behaviors: preference, feeding and learning. We show that in all cases examined the behavioral output changes in an agarose concentration-dependent manner. Our results suggest that comparisons between experiments performed on substrates differing in agarose concentration should be done with caution. It should be taken into consideration that the agarose concentration can affect the behavioral output and thereby the experimental outcomes per se potentially due to an increased escape response on more rigid substrates.

  19. An XML standard for the dissemination of annotated 2D gel electrophoresis data complemented with mass spectrometry results.

    Science.gov (United States)

    Stanislaus, Romesh; Jiang, Liu Hong; Swartz, Martha; Arthur, John; Almeida, Jonas S

    2004-01-29

    Many proteomics initiatives require a seamless bioinformatics integration of a range of analytical steps between sample collection and systems modeling immediately assessable to the participants involved in the process. Proteomics profiling by 2D gel electrophoresis to the putative identification of differentially expressed proteins by comparison of mass spectrometry results with reference databases, includes many components of sample processing, not just analysis and interpretation, are regularly revisited and updated. In order for such updates and dissemination of data, a suitable data structure is needed. However, there are no such data structures currently available for the storing of data for multiple gels generated through a single proteomic experiments in a single XML file. This paper proposes a data structure based on XML standards to fill the void that exists between data generated by proteomics experiments and storing of data. In order to address the resulting procedural fluidity we have adopted and implemented a data model centered on the concept of annotated gel (AG) as the format for delivery and management of 2D Gel electrophoresis results. An eXtensible Markup Language (XML) schema is proposed to manage, analyze and disseminate annotated 2D Gel electrophoresis results. The structure of AG objects is formally represented using XML, resulting in the definition of the AGML syntax presented here. The proposed schema accommodates data on the electrophoresis results as well as the mass-spectrometry analysis of selected gel spots. A web-based software library is being developed to handle data storage, analysis and graphic representation. Computational tools described will be made available at http://bioinformatics.musc.edu/agml. Our development of AGML provides a simple data structure for storing 2D gel electrophoresis data.

  20. An XML standard for the dissemination of annotated 2D gel electrophoresis data complemented with mass spectrometry results

    Directory of Open Access Journals (Sweden)

    Arthur John

    2004-01-01

    Full Text Available Abstract Background Many proteomics initiatives require a seamless bioinformatics integration of a range of analytical steps between sample collection and systems modeling immediately assessable to the participants involved in the process. Proteomics profiling by 2D gel electrophoresis to the putative identification of differentially expressed proteins by comparison of mass spectrometry results with reference databases, includes many components of sample processing, not just analysis and interpretation, are regularly revisited and updated. In order for such updates and dissemination of data, a suitable data structure is needed. However, there are no such data structures currently available for the storing of data for multiple gels generated through a single proteomic experiments in a single XML file. This paper proposes a data structure based on XML standards to fill the void that exists between data generated by proteomics experiments and storing of data. Results In order to address the resulting procedural fluidity we have adopted and implemented a data model centered on the concept of annotated gel (AG as the format for delivery and management of 2D Gel electrophoresis results. An eXtensible Markup Language (XML schema is proposed to manage, analyze and disseminate annotated 2D Gel electrophoresis results. The structure of AG objects is formally represented using XML, resulting in the definition of the AGML syntax presented here. Conclusion The proposed schema accommodates data on the electrophoresis results as well as the mass-spectrometry analysis of selected gel spots. A web-based software library is being developed to handle data storage, analysis and graphic representation. Computational tools described will be made available at http://bioinformatics.musc.edu/agml. Our development of AGML provides a simple data structure for storing 2D gel electrophoresis data.

  1. Agarose isoelectrofocusing of intact virions.

    Science.gov (United States)

    Zerda, K S; Gerba, C P

    1984-08-01

    A convenient and accurate method for determining the isoelectric points of intact virions is described. Tritium-labeled poliovirus 1 (strains Brunhilde and LSc-2) and echovirus 1 (isolates V239, V248, V212, R115 and 4CH-1) were successfully focused into sharp bands at their respective isoelectric points using a thin-layer agarose isoelectric focusing system. In situ detection of labeled virus bands in the agarose was by fluorography. Freezing and thawing of virus samples prior to isoelectric focusing did not alter their respective isoelectric points.

  2. Pulsed-field gel electrophoresis for Listeria monocytogenes.

    Science.gov (United States)

    Luque-Sastre, Laura; Fanning, Séamus; Fox, Edward M

    2015-01-01

    Pulsed-Field Gel Electrophoresis (PFGE) is a molecular subtyping method with high discriminatory power, reproducibility, and epidemiological concordance for the subtyping of Listeria monocytogenes and other bacteria. PFGE uses rare-cutting restriction enzymes (macrorestriction) that cut the genomic DNA, usually resulting in 6-25 DNA fragments ranging between 30 and 600 kb. Bacterial cells are immobilized in agarose plugs and subsequently lysed to release genomic DNA, which is then subjected to DNA digestion. AscI and ApaI restriction enzymes are typically used for L. monocytogenes. Electrophoresis using an alternating electric field direction results in a DNA banding pattern, or fingerprint, which is used to classify isolates into different pulsotypes. PFGE is currently the CDC's gold standard method for epidemiological studies in foodborne outbreaks.

  3. Methods of high integrity RNA extraction from cell/agarose construct.

    Science.gov (United States)

    Ogura, Takahiro; Tsuchiya, Akihiro; Minas, Tom; Mizuno, Shuichi

    2015-11-04

    Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology. Recently, molecular profiles have been obtained with extraction of a minimal volume of RNA using fluorescent-tagged quantitative polymerase chain reaction (qPCR), which requires high integrity RNA. However, the agarose interferes considerably with the quantity and quality of the extracted RNA. Moreover, little is known about RNA integrity when the RNA is extracted from cell/agarose construct. Thus, in order to obtain RNA of sufficient integrity, we examined various extraction methods and addressed reproducible methodologies for RNA extraction from cell/agarose constructs using spectrophotometry and microfluidic capillary electrophoresis. With various extraction methods using a mono-phasic solution of phenol and guanidine isothiocyanate, we evaluated quantity and quality of total RNA from cell/agarose construct. Extraction with solution of phenol and guanidine isothiocyanate followed by a silica based membrane filter column gave sufficient RNA integrity number, which allowed us to proceed to fluorescent-tagged qPCR for evaluating various cellular activities. The RNA extraction methods using phenol and guanidine isothiocyanate solution and a silica membrane column can be useful for obtaining high integrity RNA from cell/agarose constructs rich in polysaccharide and extracellular matrix. Our study contributes to further investigation using agarose hydrogels and other materials rich in polysaccharide in the field of cellular and tissue engineering.

  4. Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.

    Science.gov (United States)

    Ream, Jennifer A; Lewis, L Kevin; Lewis, Karen A

    2016-10-15

    Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5-10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Glycosaminoglycan blotting and detection after electrophoresis separation.

    Science.gov (United States)

    Volpi, Nicola; Maccari, Francesca

    2015-01-01

    Separation of glycosaminoglycans (GAGs) by electrophoresis and their characterization to the microgram level are integral parts of biochemical research. Their blotting on membranes after electrophoresis offers the advantage to perform further analysis on single separated species such as identification with antibodies and/or recovery of single band. A method for the blotting and immobilizing of several nonsulfated and sulfated complex GAGs on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose-gel electrophoresis is illustrated. This approach to the study of these complex macromolecules utilizes the capacity of agarose-gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses. Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of GAGs are capillary blotted after their separation in agarose-gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100 % and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 μg. Nonsulfated polyanions, for example hyaluronic acid (HA), may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 μg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes used for immunological detection or other applications.

  6. Denaturation and electrophoresis of RNA with formaldehyde.

    Science.gov (United States)

    Rio, Donald C

    2015-02-02

    Electrophoretic size fractionation can be used to denature and separate large mRNA molecules (0.5-10 kb) on formaldehyde-containing agarose gels. Formaldehyde contains a carbonyl group that reacts to form Schiff bases with the imino or amino groups of guanine, adenine, and cytosine. These covalent adducts prevent normal base pairing and maintain the RNA in a denatured state. Because these adducts are unstable, formaldehyde must be present in the gel to maintain the RNA in the denatured state. This protocol describes the preparation of an agarose gel with formaldehyde and its setup in a horizontal electrophoresis apparatus. RNA samples are prepared and denatured in a solution of formamide and formaldehyde and, with 0.5- to 10-kb size markers, subjected to electrophoresis through the gel. Following electrophoresis, the gel is stained to visualize RNA markers or rRNA using one of several different types of stains. © 2015 Cold Spring Harbor Laboratory Press.

  7. Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

    DEFF Research Database (Denmark)

    Boelt, Sanne Grundvad; Houen, Gunnar; Højrup, Peter

    2015-01-01

    Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation...... structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis....... measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary...

  8. Bargain Electrophoresis.

    Science.gov (United States)

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

    1986-01-01

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

  9. Hemoglobin electrophoresis

    Science.gov (United States)

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

  10. Polyacrylamide temperature gradient gel electrophoresis.

    Science.gov (United States)

    Viglasky, Viktor

    2013-01-01

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

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

    Science.gov (United States)

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

    1995-09-01

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

  12. Construction of long-range restriction maps in human DNA using pulsed field gel electrophoresis.

    Science.gov (United States)

    Gemmill, R M; Coyle-Morris, J F; McPeek, F D; Ware-Uribe, L F; Hecht, F

    1987-01-01

    Pulsed field gel electrophoresis (PFGE) is a powerful new tool for genetic analysis that can be applied to a variety of problems concerning genome structure and organization. This technique uses an agarose gel matrix to separate DNA molecules in a size range from 40 kb to 2,000 kb, molecules far larger than the maximum separable using standard agarose gel electrophoresis. The PFGE method can be used to separate the intact chromosomes from lower eukaryotes or to separate very large DNA fragments from higher eukaryotes generated by digestion with restriction endonucleases whose cleavage sites are rare. This paper describes the use of PFGE for construction of long-range restriction maps in the human genome and includes detailed methods for all steps. A pulsed field gel device that utilizes a rotating platform for altering the applied electric field is also described. Map construction is illustrated using a cloned DNA fragment (D3S2) from human chromosome 3. Several technical problems specific for mammalian genomes are discussed.

  13. Pulsed-field gel electrophoresis (PFGE): application in population structure studies of bovine mastitis-causing streptococci.

    Science.gov (United States)

    Santos-Sanches, Ilda; Chambel, Lélia; Tenreiro, Rogério

    2015-01-01

    Pulsed-field gel electrophoresis (PFGE) separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens and is a valuable typing scheme to detect and differentiate strains. Particularly, the contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of many bacterial pathogens. Here we describe a PFGE protocol that was applicable to the study of bovine streptococci, namely, Streptococcus agalactiae (group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (group C Streptococcus, GCS), and Streptococcus uberis-which are relevant pathogens causing mastitis, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production.

  14. Synthesis of agarose-metal/semiconductor nanoparticles having ...

    Indian Academy of Sciences (India)

    Agarose, a naturally occurring biopolymer is used for the stabilization of metal, semiconductor nanoparticles. Ag and Cu nanoparticles stabilized in agarose matrix show excellent antibacterial activity against E. coli bacteria. The well dispersed metal nanoparticles within the agarose composite films can be readily converted ...

  15. Improved DNA Electrophoresis in Conditions Favoring Polyborates and Lewis Acid Complexation

    OpenAIRE

    Singhal, Hari; Ren, Yunzhao R.; Kern, Scott E.

    2010-01-01

    Spatial compression among the longer DNA fragments occurs during DNA electrophoresis in agarose and non-agarose gels when using certain ions in the conductive buffer, impairing the range of fragment sizes resolved well in a single gel. Substitutions using various polyhydroxyl anions supported the underlying phenomenon as the complexation of Lewis acids to DNA. We saw significant improvements using conditions (lithium borate 10 mM cations, pH 6.5) favoring the formation of borate polyanions an...

  16. Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression

    Directory of Open Access Journals (Sweden)

    Mallein-Gerin Frédéric

    2008-09-01

    Full Text Available Abstract Background Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice. Results Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C™ Flexercell® Compression Plus™ System (Flexcell. After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the

  17. Polyacrylamide gel electrophoresis of RNA.

    Science.gov (United States)

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W

    2010-06-01

    Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. This technique is generally applicable for RNA detection, quantification, purification by size, and quality assessment. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to RNA-protein complexes, native gels are appropriate. The only disadvantage to acrylamide gels is that they are not suitable for analyzing large RNAs (> or =600 nt); for such applications, agarose gels are preferred. This protocol describes how to prepare, load, and run polyacrylamide gels for RNA analysis.

  18. Crosslinking of agarose bioplastic using citric acid.

    Science.gov (United States)

    Awadhiya, Ankur; Kumar, David; Verma, Vivek

    2016-10-20

    We report chemical crosslinking of agarose bioplastic using citric acid. Crosslinking was confirmed using Fourier transform infrared (FTIR) spectroscopy. The effects of crosslinking on the tensile strength, swelling, thermal stability, and degradability of the bioplastic were studied in detail. The tensile strength of the bioplastic films increased from 25.1MPa for control films up to a maximum of 52.7MPa for citric acid crosslinked films. At 37°C, the amount of water absorbed by crosslinked agarose bioplastic was only 11.5% of the amount absorbed by non-crosslinked controls. Thermogravimetric results showed that the crosslinked samples retain greater mass at high temperature (>450°C) than control samples. Moreover, while the crosslinked films were completely degradable, the rate of degradation was lower compared to non-crosslinked controls. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. DNA ELECTROPHORESIS AT SURFACES

    Energy Technology Data Exchange (ETDEWEB)

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  20. Implementation of USP antibody standard for system suitability in capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for release and stability methods.

    Science.gov (United States)

    Esterman, Abbie L; Katiyar, Amit; Krishnamurthy, Girija

    2016-09-05

    Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is widely used for purity analysis of monoclonal antibody therapeutics for release and stability to demonstrate product consistency and shelf life during the manufacturing and life cycle of the product. CE-SDS method development is focused on exploring the method capability to provide the information about the product purity and product related degradants (fragmentation, aggregation etc.). In order to establish the functionality of the instrumentation, software, and sample preparation; system suitability criteria need to be defined for analytical methods using a well characterized reference standard run under the same protocol and analysis as the test articles. Typically the reference standard is produced using a manufacturing process representative of the clinical material. The qualification, control, and maintenance of in-house reference standards are established through rigorous quality and regulatory guidelines. The U.S. Pharmacopeia (USP) has developed a monoclonal IgG System Suitability Reference Standard to be utilized for assessment of system suitability in CE-SDS methods. In this communication, we evaluate the system suitability acceptance criteria performance of the USP IgG standard using two methods, the recommended USP protocol provided in monograph and a molecule specific Bristol-Myers Squibb (BMS) CE-SDS method. The results from USP IgG standard were compared with two in-house monoclonal antibody reference standards. The data suggest that the USP CE-SDS method may not be suitable for CE-SDS analysis for release and stability of monoclonal antibody therapeutics due to the high level of method induced partial reduction observed for all molecules tested. This high level of fragmentation observed utilizing the USP method will result in reporting lower purity levels, which will impact the overall quality assessment of the molecule. The system suitability criteria recommended by the USP method can be

  1. Presence of a novel DNA methylation enzyme in methicillin-resistant Staphylococcus aureus isolates associated with pig farming leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis.

    NARCIS (Netherlands)

    Bens, C.C.; Voss, A.; Klaassen, C.H.W.

    2006-01-01

    Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in

  2. Presence of a Novel DNA Methylation Enzyme in Methicillin-Resistant Staphylococcus aureus Isolates Associated with Pig Farming Leads to Uninterpretable Results in Standard Pulsed-Field Gel Electrophoresis Analysis

    OpenAIRE

    Bens, Corina C. P. M.; Voss, Andreas; Klaassen, Corné H. W.

    2006-01-01

    Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in the genus Staphylococcus with the recognition sequence CCNGG.

  3. Cobalt complexes as internal standards for capillary zone electrophoresis-mass spectrometry studies in biological inorganic chemistry.

    Science.gov (United States)

    Holtkamp, Hannah U; Morrow, Stuart J; Kubanik, Mario; Hartinger, Christian G

    2017-07-01

    Run-by-run variations are very common in capillary electrophoretic (CE) separations and cause imprecision in both the migration times and the peak areas. This makes peak and kinetic trend identification difficult and error prone. With the aim to identify suitable standards for CE separations which are compatible with the common detectors UV, ESI-MS, and ICP-MS, the Co III complexes [Co(en) 3 ]Cl 3 , [Co(acac) 3 ] and K[Co(EDTA)] were evaluated as internal standards in the reaction of the anticancer drug cisplatin and guanosine 5'-monophosphate as an example of a classical biological inorganic chemistry experiment. These Co III chelate complexes were considered for their stability, accessibility, and the low detection limit for Co in ICP-MS. Furthermore, the Co III complexes are positively and negatively charged as well as neutral, allowing the detection in different areas of the electropherograms. The background electrolytes were chosen to cover a wide pH range. The compatibility to the separation conditions was dependent on the ligands attached to the Co III centers, with only the acetylacetonato (acac) complex being applicable in the pH range 2.8-9.0. Furthermore, because of being charge neutral, this compound could be used as an electroosmotic flow (EOF) marker. In general, employing Co complexes resulted in improved data sets, particularly with regard to the migration times and peak areas, which resulted, for example, in higher linear ranges for the quantification of cisplatin.

  4. Denaturation and electrophoresis of RNA with glyoxal.

    Science.gov (United States)

    Rio, Donald C

    2015-02-02

    This protocol is used to denature and separate large mRNA molecules (0.5-10 kb) on agarose gels by electrophoretic size fractionation. Glyoxal (also called diformyl or ethanedial), the agent responsible for maintaining denaturation in this protocol, contains two carbonyl groups that react to form a cyclic ring structure with the imino and amino groups of guanine. It can also react with the amino groups of adenine and cytidine. When RNA is denatured in the presence of glyoxal, this covalent adduct prevents normal base pairing and maintains the RNA in a denatured state in agarose gels. Once formed, these adducts are stable at room temperature at pH RNA in the denatured state. Because the fully denatured RNA migrates through agarose gels according to its molecular mass, this method can be used to accurately size mRNA molecules. Following electrophoresis and reversal of glyoxalation, the RNA can be detected using a northern hybridization procedure. © 2015 Cold Spring Harbor Laboratory Press.

  5. Comparison of the separation of nine tryptamine standards based on gas chromatography, high performance liquid chromatography and capillary electrophoresis methods.

    Science.gov (United States)

    Wang, Man-Juing; Liu, Ju-Tsung; Chen, Hung-Ming; Lin, Jian-Jhih; Lin, Cheng-Huang

    2008-02-15

    Nine tryptamines, including alpha-methyltryptamine (AMT), N,N-dimethyltryptamine (DMT), 5-methoxy-alpha-methyltryptamine (5-MeO-AMT), N,N-diethyltryptamine (DET), N,N-dipropyltryptamine (DPT), N,N-dibutyltryptamine (DBT), N,N-diisopropyltryptamine (DIPT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) were selected as model compounds. Comparisons of their sensitivity, selectivity, time, cost and the order of migration are described based on different separation techniques (GC, HPLC and CE, respectively). As a result, the limit of detection (S/N=3) obtained by GC/MS and LC/UV-absorption ranged from 0.5 to 15 microg/mL and 0.3 to 1.0 microg/mL, respectively. In contrast to this, based on the CZE/UV-absorption method, the limit of detection (S/N=3) was determined to 0.5-1 microg/mL. However, when the sweeping-MEKC mode was applied, it dramatically improved to 2-10 ng/mL. In the case of GC, HPLC and CE, migration times of the nine standards ranged from 11 to 15 min and 8 to 23 min by GC and HPLC, respectively; ranged from 20 to 26 min by sweeping-MEKC. The order of migration of DMT, DET, DPT and DBT follows the molecular weight, whereas the order of migration of AMT and 5-MeO-AMT (primary amines), DIPT (an isomer of DPT) and 5-methoxy-tryptamines (5-MeO-AMT, 5-MeO-DMT and 5-MeO-DIPT) can be altered by changing the separation conditions.

  6. 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.

  7. Understanding Electrophoresis through the Investigation of Size, Shape, and Charge of pH Indicators

    Science.gov (United States)

    Brenner, Ryan K.; Hess, Kenneth R.; Morford, Jennifer L.

    2015-01-01

    A laboratory experiment was designed for upper-level students in a Chemical Analysis course to illustrate the theoretical and practical applications of 0.8% agarose gel electrophoresis and to reinforce an understanding of weak acids/bases using easy-to-visualize pH indicators. The careful choice of indicators included acid and base types with…

  8. Potential of Agarose/Silk Fibroin Blended Hydrogel for in Vitro Cartilage Tissue Engineering.

    Science.gov (United States)

    Singh, Yogendra Pratap; Bhardwaj, Nandana; Mandal, Biman B

    2016-08-24

    An osteoarthritis pandemic has accelerated exploration of various biomaterials for cartilage reconstruction with a special emphasis on silk fibroin from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk worms. Retention of positive attributes of the agarose standard and nullification of its negatives are central to the current agarose/silk fibroin hydrogel design. In this study, hydrogels of mulberry and non-mulberry silk fibroin blended with agarose were fabricated and evaluated in vitro for two weeks for cartilaginous tissue formation. The fabricated hydrogels were physicochemically characterized and analyzed for cell viability, proliferation, and extra cellular matrix deposition. The amalgamation of silk fibroin with agarose impacted the pore size, as illustrated by field emission scanning electron microscopy studies, swelling behavior, and in vitro degradation of the hydrogels. Fourier transform infrared spectroscopy results indicated the blend formation and confirmed the presence of both components in the fabricated hydrogels. Rheological studies demonstrated enhanced elasticity of blended hydrogels with G' > G″. Biochemical analysis revealed significantly higher levels of sulfated glycosaminoglycans (sGAGs) and collagen (p ≤ 0.01) in blended hydrogels. More specifically, the non-mulberry silk fibroin blend showed sGAG and collagen content (∼1.5-fold) higher than that of the mulberry blend (p ≤ 0.05). Histological and immunohistochemical analyses further validated the enhanced deposition of sGAG and collagen, indicating maintenance of chondrogenic phenotype within constructs after two weeks of culture. Real-time PCR analysis further confirmed up-regulation of cartilage-specific aggrecan, sox-9 (∼1.5-fold) and collagen type II (∼2-fold) marker genes (p ≤ 0.01) in blended hydrogels. The hydrogels demonstrated immunocompatibility, which was evidenced by minimal in vitro secretion of tumor necrosis factor-α (TNF-α) by murine

  9. An external standard method for quantification of human cytomegalovirus by PCR

    International Nuclear Information System (INIS)

    Rongsen, Shen; Liren, Ma; Fengqi, Zhou; Qingliang, Luo

    1997-01-01

    An external standard method for PCR quantification of HCMV was reported. [α- 32 P]dATP was used as a tracer. 32 P-labelled specific amplification product was separated by agarose gel electrophoresis. A gel piece containing the specific product band was excised and counted in a plastic scintillation counter. Distribution of [α- 32 P]dATP in the electrophoretic gel plate and effect of separation between the 32 P-labelled specific product and free [α- 32 P]dATP were observed. A standard curve for quantification of HCMV by PCR was established and detective results of quality control templets were presented. The external standard method and the electrophoresis separation effect were appraised. The results showed that the method could be used for relative quantification of HCMV. (author)

  10. SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

    OpenAIRE

    Kiltie, A E; Ryan, A J

    1997-01-01

    Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This meth...

  11. Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

    Science.gov (United States)

    Tomizawa, M; Latli, B; Casida, J E

    1996-10-01

    Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

  12. Agarose gel electrophoretic detection of six beta-lactam antibiotic residues in milk.

    Science.gov (United States)

    Cutting, J H; Kiessling, W M; Bond, F L; McCarron, J E; Kreuzer, K S; Hurlbut, J A; Sofos, J N

    1995-01-01

    An electrophoretic method coupled with bioautography was developed for detection and identification of penicillin G, ampicillin, amoxicillin, cloxacillin, cephapirin, and ceftiofur residues in milk. The method uses a 2% agarose gel for electrophoresis, an overlay of PM indicator agar seeded with Bacillus stearothermophilus var. calidolactis, and incubation at 55 degrees C for 16-18 h. The new method separated and detected residues in milk at the levels of concern for the Food and Drug Administration (FDA) for penicillin G (5 ppb), cephapirin (20 ppb), and ceftiofur (50 ppb). The method also detected ampicillin, amoxicillin, and cloxacillin at 20, 30, and 30 ppb, respectively, but these levels are above those of concern for FDA (10 ppb).

  13. Protein electrophoresis - urine

    Science.gov (United States)

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

  14. Serum globulin electrophoresis

    Science.gov (United States)

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

  15. Uptake and Recovery of Lead by Agarose Gel Polymers

    OpenAIRE

    Anurag Pandey; Anupam Shukla; Lalitagauri Ray

    2009-01-01

    Problem statement: The uptake and recovery of lead ions were investigated by using agarose gel polymers. Approach: The experimental results showed that the agarose gel were effective in removing Pb (II) from solution. Biosorption equilibrium was approached within 4 h. Pseudo second-order was applicable to all the sorption data over the entire time range. Results: The sorption data conformed well to both the Langmuir and the Freundlich isotherm model. The ma...

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  17. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    OpenAIRE

    Hubbard Alan E; Dorsey Grant; Gupta Vinay; Rosenthal Philip J; Greenhouse Bryan

    2010-01-01

    Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary elec...

  18. 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)

  19. 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.

  20. Passivated gel electrophoresis of charged nanospheres by light-scattering video tracking.

    Science.gov (United States)

    Zhu, Xiaoming; Mason, Thomas G

    2014-08-15

    Gel electrophoresis (gel-EP) has been used for decades to separate charged biopolymers, such as DNA, RNA, and proteins, yet propagation of other charged colloidal objects, such as nanoparticles, during gel-EP has been studied comparatively little. Simply introducing anionic nanoparticles, such as sulfate-stabilized polystyrene nanospheres, in standard large-pore agarose gels commonly used for biomolecules does not automatically ensure propagation or size-separation because attractive interactions can exist between the gel and the nanoparticles. Whereas altering the surfaces of the nanoparticles is a possible solution, here, by contrast, we show that treating a common type I-A low-electroendoosmosis agarose gel with a passivation agent, such as poly-(ethyleneglycol), enables charged nanoparticles to propagate through large-pore passivated gels in a highly reproducible manner. Moreover, by taking advantage of the significant optical scattering from the nanoparticles, which is not easily measurable for biopolymers, relative to scattering from the gel, we perform real-time, light-scattering, video-tracking gel-EP. Continuous optical measurements of the propagation of bands of uniformly sized nanospheres in passivated gels provides the propagation distance, L, and velocity, v, as a function of time for different sphere radii, electric field strengths, gel concentrations, and passivation agent concentrations. The steady-state particle velocities vary linearly with applied electric field strength, E, for small E, but these velocities become non-linear for larger E, suggesting that strongly driven nanoparticles can become elastically trapped in the smaller pores of the gel, which act like blind holes, in a manner that thermal fluctuations cannot overcome. Based on this assumption, we introduce a simple model that fits the measured v(E) in both linear and non-linear regimes over a relevant range of applied voltages. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Nonlinear electrophoresis for purification of soil DNA for metagenomics.

    Science.gov (United States)

    Engel, Katja; Pinnell, Lee; Cheng, Jiujun; Charles, Trevor C; Neufeld, Josh D

    2012-01-01

    Purification of microbial DNA from soil is challenging due to the co-extraction of humic acids and associated phenolic compounds that inhibit subsequent cloning, amplification or sequencing. Removal of these contaminants is critical for the success of metagenomic library construction and high-throughput sequencing of extracted DNA. Using three different composite soil samples, we compared a novel DNA purification technique using nonlinear electrophoresis on the synchronous coefficient of drag alteration (SCODA) instrument with alternate purification methods such as direct current (DC) agarose gel electrophoresis followed by gel filtration or anion exchange chromatography, Wizard DNA Clean-Up System, and the PowerSoil DNA Isolation kit. Both nonlinear and DC electrophoresis were effective at retrieving high-molecular weight DNA with high purity, suitable for construction of large-insert libraries. The PowerSoil DNA Isolation kit and the nonlinear electrophoresis had high recovery of high purity DNA suitable for sequencing purposes. All methods demonstrated high consistency in the bacterial community profiles generated from the DNA extracts. Nonlinear electrophoresis using the SCODA instrument was the ideal methodology for the preparation of soil DNA samples suitable for both high-throughput sequencing and large-insert cloning applications. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. 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...

  3. Exactly solvable Ogston model of gel electrophoresis. IX. Generalizing the lattice model to treat high field intensities

    Science.gov (United States)

    Gauthier, Michel G.; Slater, Gary W.

    2002-10-01

    Traditionally, the Ogston regime is studied solely in the limit of low field intensities. This explains why the theoretical discussion has focused until now on the relative roles of the fractional volume available to the analyte and the subtleties of the gel architecture. Over the past several years, we have developed a lattice model of gel electrophoresis that has allowed us to revisit the fundamental assumptions of the standard Ogston model. In particular, we demonstrated that the fractional free volume is not the relevant parameter for gel sieving. In this article, we continue the development of this model and we generalize our mathematical approach to treat nonvanishing electric field intensities. To do so, we must revisit the way biased random walks are normally modeled by stochastic processes. Straightforward generalizations based on standard Metropolis-like schemes fail at high field intensities. Moreover, our generalization requires the complete decoupling of the spatial directions parallel and perpendicular to the field direction. We show that our novel theoretical approach makes it possible to calculate exact mobilities in the presence of lattice obstacles. Several two-dimensional examples are then studied, including one that includes topological dead ends that act like traps. In the latter case, we recover results very similar to those reported by Serwer et al. [Biopolymers 29, 1863 (1990)] on the trapping electrophoresis of charged spheres in agarose gels. In the absence of such traps, the mobility is shown to be a very weak function of the electric field, thus validating the historical neglect of the field intensity in the development of obstruction models for the Ogston sieving regime of small analytes. Finally, we describe how the present model could be improved to treat more realistic cases and we discuss the problem of the field dependence of the diffusion coefficient during electrophoresis.

  4. Synthesis of agarose-metal/semiconductor nanoparticles having ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 120; Issue 6. Synthesis of agarose-metal/semiconductor nanoparticles having superior bacteriocidal activity and their simple conversion to metal-carbon composites. K K R Datta B Srinivasan H Balaram M Eswaramoorthy. Volume 120 Issue 6 November 2008 pp 579- ...

  5. Diffusion properties of bacteriophages through agarose gel membrane.

    Science.gov (United States)

    Hu, Jun; Miyanaga, Kazuhiko; Tanji, Yasunori

    2010-01-01

    A simple two-chamber diffusion method was developed to study the diffusion properties of bacteriophages (phages). The apparent diffusion coefficients (D(app)) of Myoviridae phage T4 and filamentous phage fNEL were investigated, and the diffusion of the phages was found to be much slower than the diffusion of three antibiotics, ciprofloxacin, penicillin G, and tetracycline. D(app) of T4 and fNEL in water through filter paper were calculated to be 2.8 x 10⁻¹¹ m²/s and 6.8 x 10⁻¹² m²/s, respectively, and D(app) of fNEL through agarose gel membrane, an artificial biofilm, was also calculated to be smaller than that of T4. In addition, D(app) of phages through agarose gel was dependent on agarose concentration due to the similar size of phage and agarose gel mesh. We concluded that D(app) of phages through an artificial biofilm is dependent on both phage morphology and biofilm density, and suggest the use of this method to study diffusion properties through real biofilms. © 2010 American Institute of Chemical Engineers

  6. Recovery of DNA from agarose gel by trap method

    African Journals Online (AJOL)

    Administrator

    2011-09-05

    Sep 5, 2011 ... target band and refilling it with the same buffer. When the molecules of DNA move from agarose gel into a well, the velocity of DNA will increase in the well without interaction between gel and DNA (Stellwagen and. Stellwagen, 2009). It is possible to recover them from the. *Corresponding author. E-mail: ...

  7. 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.

  8. 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 ...

  9. Multilaboratory validation study of standardized multiple-locus variable-number tandem repeat analysis protocol for shiga toxin-producing Escherichia coli O157: a novel approach to normalize fragment size data between capillary electrophoresis platforms.

    Science.gov (United States)

    Hyytia-Trees, Eija; Lafon, Patricia; Vauterin, Paul; Ribot, Efrain M

    2010-02-01

    The PulseNet USA subtyping network recently established a standardized protocol for multiple-locus variable-number tandem repeat analysis (MLVA) to characterize Shiga toxin-producing Escherichia coli O157. To enable data comparisons from different laboratories in the same database, reproducibility and high quality of the data must be ensured. The aim of this study was to test the robustness and reproducibility of the proposed standardized protocol by subjecting it to a multilaboratory validation process and to address any discrepancies that may have arisen from the study. A set of 50 strains was tested in 10 PulseNet participating laboratories that used capillary electrophoresis instruments from two manufacturers. Six out of the 10 laboratories were able to generate correct MLVA types for 46 (92%) or more strains. The discrepancies in MLVA type assignment were caused mainly by difficulties in optimizing polymerase chain reactions that were attributed to technical inexperience of the staff and suboptimal quality of reagents and instrumentation. It was concluded that proper training of staff must be an integral part of technology transfer. The interlaboratory reproducibility of fragment sizing was excellent when the same capillary electrophoresis platform was used. However, sizing discrepancies of up to six base pairs for the same fragment were detected between the two platforms. These discrepancies were attributed to different dye and polymer chemistries employed by the manufacturers. A novel software script was developed to assign alleles based on two platform-specific (Beckman Coulter CEQ8000 and Applied Biosystems Genetic Analyzer 3130xl) look-up tables containing fragment size ranges for all alleles. The new allele assignment method was validated at the PulseNet central laboratory using a diverse set of 502 Shiga toxin-producing Escherichia coli O157 isolates. The validation confirmed that the script reliably assigned the same allele for the same fragment

  10. SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

    Science.gov (United States)

    Kiltie, A E; Ryan, A J

    1997-07-15

    Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This method is as sensitive as [3H]thymidine prelabelling of cells and allows DNA dsb to be measured simply and economically in non-cycling cells.

  11. Analysis of rRNA gene methylation in Arabidopsis thaliana by CHEF-Conventional 2D gel electrophoresis

    Science.gov (United States)

    Mohannath, Gireesha; Pikaard, Craig S.

    2017-01-01

    Summary Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb to 9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes and sub-chromosomal DNA fragments, etc. Here we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~ 4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes. PMID:27576719

  12. Agarose and methylcellulose hydrogel blends for nerve regeneration applications

    Science.gov (United States)

    Martin, Benton C.; Minner, Eric J.; Wiseman, Sherri L.; Klank, Rebecca L.; Gilbert, Ryan J.

    2008-06-01

    Trauma sustained to the central nervous system is a debilitating problem for thousands of people worldwide. Neuronal regeneration within the central nervous system is hindered by several factors, making a multi-faceted approach necessary. Two factors contributing to injury are the irregular geometry of injured sites and the absence of tissue to hold potential nerve guides and drug therapies. Biocompatible hydrogels, injectable at room temperature, that rapidly solidify at physiological temperatures (37 °C) are beneficial materials that could hold nerve guidance channels in place and be loaded with therapeutic agents to aid wound healing. Our studies have shown that thermoreversible methylcellulose can be combined with agarose to create hydrogel blends that accommodate these properties. Three separate novel hydrogel blends were created by mixing methylcellulose with one of the three different agaroses. Gelation time tests show that the blends solidify at a faster rate than base methylcellulose at 37 °C. Rheological data showed that the elastic modulus of the hydrogel blends rapidly increases at 37 °C. Culturing experiments reveal that the morphology of dissociated dorsal root ganglion neurons was not altered when the hydrogels were placed onto the cells. The different blends were further assessed using dissolution tests, pore size evaluations using scanning electron microscopy and measuring the force required for injection. This research demonstrates that blends of agarose and methylcellulose solidify much more quickly than plain methylcellulose, while solidifying at physiological temperatures where agarose cannot. These hydrogel blends, which solidify at physiological temperatures naturally, do not require ultraviolet light or synthetic chemical cross linkers to facilitate solidification. Thus, these hydrogel blends have potential use in delivering therapeutics and holding scaffolding in place within the nervous system.

  13. Development of rocket electrophoresis technique as an analytical tool in preformulation study of tetanus vaccine formulation.

    Science.gov (United States)

    Ahire, V J; Sawant, K K

    2006-08-01

    Rocket Electrophoresis (RE) technique relies on the difference in charges of the antigen and antibodies at the selected pH. The present study involves optimization of RE run conditions for Tetanus Toxoid (TT). Agarose gel (1% w/v, 20 ml, pH 8.6), anti-TT IgG - 1 IU/ml, temperature 4-8 degrees C and run duration of 18 h was found to be optimum. Height of the rocket-shaped precipitate was proportional to TT concentration. The RE method was found to be linear in the concentration range of 2.5 to 30 Lf/mL. The method was validated and found to be accurate, precise, and reproducible when analyzed statistically using student's t-test. RE was used as an analytical method for analyzing TT content in plain and marketed formulations as well as for the preformulation study of vaccine formulation where formulation additives were tested for compatibility with TT. The optimized RE method has several advantages: it uses safe materials, is inexpensive, and easy to perform. RE results are less prone to operator's bias as compared to flocculation test and can be documented by taking photographs and scanned by densitometer; RE can be easily standardized for the required antigen concentration by changing antitoxin concentration. It can be used as a very effective tool for qualitative and quantitative analysis and in preformulation studies of antigens.

  14. Nonwoven Carboxylated Agarose-Based Fiber Meshes with Antimicrobial Properties.

    Science.gov (United States)

    Forget, Aurelien; Arya, Neha; Randriantsilefisoa, Rotsiniaina; Miessmer, Florian; Buck, Marion; Ahmadi, Vincent; Jonas, Daniel; Blencowe, Anton; Shastri, V Prasad

    2016-12-12

    Hydrogel forming polysaccharides, such as the seaweed derived agarose, are well suited for wound dressing applications as they have excellent cell and soft tissue compatibility. For wound dressings, fibrous structure is desirable as the high surface area can favor adsorption of wound exudate and promote drug delivery. Although electrospinning offers a straightforward means to produce nonwoven fibrous polymeric structures, processing agarose and its derivatives into fibers through electrospinning is challenging as it has limited solubility in solvents other than water. In this study we describe the processing of carboxylated agarose (CA) fibers with antibacterial properties by electrospinning from a solution of the ionic liquid (IL) 1-butyl-3-methylimidazolium chloride ([Bmim] + Cl - ) possessing antimicrobial properties. The extent of carboxylation was found to impact fiber diameter, mesh elastic modulus, fiber swelling, and the loading and release of IL. IL-bearing CA fibers inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa, bacteria commonly found in wound exudate. In sum, nonwoven CA fibers processed from IL are promising as biomaterials for wound dressing applications.

  15. Preparation and stability of agarose microcapsules containing BCG.

    Science.gov (United States)

    Esquisabel, A; Hernandez, R M; Igartua, M; Gascón, A R; Calvo, B; Pedraz, J L

    2002-01-01

    An emulsification/internal gelation method of preparing small-sized agarose microcapsules containing Bacillus Calmette-Guerin (BCG) is reported. Agarose microcapsules have been prepared by the emulsification of the hydrogel within a vegetable oil followed by its gelation due to the cooling of the system. Four different oils (sesame, sweet almonds, camomile and jojoba) were assayed. The rheological analysis of the oils showed a Newtonian behaviour, with viscosity values of 37.7, 51.2, 59.3 and 67.1 mPa s for jojoba, camomile, sesame and sweet almonds oil, respectively. The particle size of the microcapsules obtained ranged from 23.1 microm for the microcapsules prepared with sweet almonds oil to 42.6 microm for those prepared with jojoba. The microcapsule particle size was found to be dependent on the viscosity of the oil used in the emulsification step. The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Once prepared, microcapsules were freeze-dried using 5% trehalose as cryoprotectant and the stability of the microcapsules was assayed during 12 months storage at room temperature, observing that agarose microcapsules were stable after 12 months storage, since there was no evidence of alteration in the freeze-dried appearance, resuspension rate, observation under microscope, or particle size.

  16. Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A Biophysical Chemistry Laboratory Experiment

    Science.gov (United States)

    Shafirovich, Vladimir; Singh, Carolyn; Geacintov, Nicholas E.

    2003-11-01

    Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. This article describes the design of several simple experiments to explore DNA damage initiated by photoinduced electron-transfer reactions sensitized by the acridine derivative, proflavine (PF). A supercoiled DNA agarose gel nicking assay is employed as a sensitive probe of DNA strand cleavage. A low-cost experimental and computer-interfaced imaging apparatus is described allowing for the digital recording and analysis of the gel electrophoresis results. The first experiment describes the formation of direct strand breaks in double-stranded DNA induced by photoexcitation of the intercalated PF molecules. The second experiment demonstrates that the addition of the well-known electron acceptor, methylviologen, gives rise to a significant enhancement of the photochemical DNA strand cleavage effect. This occurs by an electron transfer step to methylviologen that renders the inital photoinduced charge separation between photoexcited PF and DNA irreversible. The third experiment demonstrates that the action spectrum of the DNA photocleavage matches the absorption spectrum of DNA-bound, intercalated PF molecules, which differs from that of free PF molecules. This result demonstrates that the photoinduced DNA strand cleavage is initiated by intercalated rather than free PF molecules.

  17. Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds

    Science.gov (United States)

    Karasawa, Takatoshi; Sibrian-Vazquez, Martha; Strongin, Robert M.; Steyger, Peter S.

    2013-01-01

    Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment. PMID:23755301

  18. Identification of cisplatin-binding proteins using agarose conjugates of platinum compounds.

    Directory of Open Access Journals (Sweden)

    Takatoshi Karasawa

    Full Text Available Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94, heat shock protein 90 (HSP90, calreticulin, valosin containing protein (VCP, and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

  19. The distribution of particles characterized by size and free mobility within polydisperse populations of protein-polysaccharide conjugates, determined from two-dimensional agarose electropherograms.

    Science.gov (United States)

    Tietz, D; Aldroubi, A; Schneerson, R; Unser, M; Chrambach, A

    1991-01-01

    New approaches for the characterization of polydisperse particle populations are presented*. The investigated samples contain virus-sized protein-polysaccharide conjugates which had previously been prepared as immunogens against bacterial meningitis (Hib). The analysis is based on two-dimensional agarose electrophoresis (Serwer-type). This method, like the one of O'Farrell, achieves a separation according to size and charge. It relies on a different principle, however, and is applicable to nondenatured particles which are 100 to more than 1000 times larger in mass than regular uncrosslinked proteins. Data from stained gel patterns are evaluated by the computer program ELPHOFIT, which makes it possible to standardize the gel and to construct a nomogram which defines every position on the gel in terms of particle size and free mobility (related to surface net charge density). The output of ELPHOFIT, consisting of nomogram parameters, is transferred to the image processing program GELFIT. This software is used to evaluate the computer images obtained by digitizing the stained gel patterns: (i) The nomogram is electronically superimposed on the computer image. (ii) The gel pattern is transformed from a curvilinear to a rectangular coordinate system of particle size and free mobility. The center of gravity as well as density maxima are given in coordinates of particle size and free mobility. Ranges of grey levels can be accentuated by adding 16 pseudocolors. (iii) Using surface-stripping techniques, GELFIT provides an estimate for the number of major subpopulations within each preparation. (iv) Numerical values for the distribution of particle size and free mobility are determined. Using program IMAGE, the quantitative physical assessment of a given conjugate preparation is presented in the form of a computer-generated three-dimensional plot, the shape of which serves to identify and characterize the preparation visually. The data analysis based on digitized two

  20. Analysis of serum transthyretin by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using magnetic beads.

    Science.gov (United States)

    Peró-Gascón, Roger; Pont, Laura; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2016-05-01

    In this paper, an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using magnetic beads (MBs) is described for the analysis of serum transthyretin (TTR), which is a protein related to different types of amyloidosis. First, purification of TTR from serum was investigated by off-line immunoprecipitation and CE-MS. The suitability of three Protein A (ProA) MBs (Protein A Ultrarapid Agarose(TM) (UAPA), Dynabeads(®) Protein A (DyPA) and SiMAG-Protein A (SiPA) and AffiAmino Ultrarapid Agarose(TM) (UAAF) MBs to prepare an IA sorbent with a polyclonal antibody (Ab) against TTR, was studied. In all cases, results were repeatable and it was possible the identification and the quantitation of the relative abundance of the six most abundant TTR proteoforms. Although recoveries were the best with UAPA MBs, UAAF MBs were preferred for on-line immunopurification because Ab was not eluted from the MBs. Under the optimized conditions with standards in IA-SPE-CE-MS, microcartridge lifetime (>20 analyses/day) and repeatability (2.9 and 4.3% RSD for migration times and peak areas) were good, the method was linear between 5 and 25 μg/mL and LOD was around 1 μg/mL (25 times lower than by CE-MS, ≈25 μg/mL). A simple off-line sample pretreatment based on precipitation of the most abundant proteins with 5% (v/v) of phenol was necessary to clean-up serum samples. The potential of the on-line method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was demonstrated analysing serum samples from healthy controls and FAP-I patients. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Boronate affinity saccharide electrophoresis: a novel carbohydrate analysis tool.

    Science.gov (United States)

    Jackson, Thomas R; Springall, Jeremy S; Rogalle, Damien; Masumoto, Naoko; Ching Li, Hung; D'Hooge, François; Perera, Semali P; Jenkins, A Toby A; James, Tony D; Fossey, John S; van den Elsen, Jean M H

    2008-11-01

    The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore-assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5-1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar-containing molecules.

  2. Capillary electrophoresis of diuretics.

    Science.gov (United States)

    Riekkola, M L; Jumppanen, J H

    1996-05-31

    The review surveys the application of capillary electrophoresis to the screening, identification and determination of diuretics and probenecid. The number of publications is still limited, but the studies already published clearly show that capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography are excellent alternatives for the investigation of diuretics. High accuracy identifications of diuretics and probenecid, even in urine samples, can be obtained when CZE is used with the marker techniques. This review paper has been written from the viewpoint of practical use and some hints are given for future CE studies on diuretics.

  3. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    Science.gov (United States)

    2010-01-01

    Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL) vs. dihydroartemisinin-piperaquine (DP) performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes. Results Capillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66) and poor agreement in Apac (kappa = 0.24). Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5). However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03). Risk differences between AL and DP were not significantly different whether gel or capillary methods were used. Conclusions Genotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission, gel electrophoresis

  4. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda.

    Science.gov (United States)

    Gupta, Vinay; Dorsey, Grant; Hubbard, Alan E; Rosenthal, Philip J; Greenhouse, Bryan

    2010-01-15

    Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Samples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL) vs. dihydroartemisinin-piperaquine (DP) performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes. Capillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66) and poor agreement in Apac (kappa = 0.24). Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5). However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03). Risk differences between AL and DP were not significantly different whether gel or capillary methods were used. Genotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission, gel electrophoresis appears adequate to estimate comparative

  5. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    Directory of Open Access Journals (Sweden)

    Hubbard Alan E

    2010-01-01

    Full Text Available Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL vs. dihydroartemisinin-piperaquine (DP performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes. Results Capillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66 and poor agreement in Apac (kappa = 0.24. Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5. However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03. Risk differences between AL and DP were not significantly different whether gel or capillary methods were used. Conclusions Genotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission

  6. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    International Nuclear Information System (INIS)

    Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de

    2008-01-01

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl 2 ) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl 2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl 2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  7. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis.

    Science.gov (United States)

    Schwartz, D C; Cantor, C R

    1984-05-01

    A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.

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

    Science.gov (United States)

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

    1992-04-01

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

  9. Structural aspects of magnetic fluid stabilization in aqueous agarose solutions

    Science.gov (United States)

    Nagornyi, A. V.; Petrenko, V. I.; Avdeev, M. V.; Yelenich, O. V.; Solopan, S. O.; Belous, A. G.; Gruzinov, A. Yu.; Ivankov, O. I.; Bulavin, L. A.

    2017-06-01

    Structure characterization of magnetic fluids (MFs) synthesized by three different methods in aqueous solutions of agarose was done by means of small-angle neutron (SANS) and synchrotron X-ray scattering (SAXS). The differences in the complex aggregation observed in the studied magnetic fluids were related to different stabilizing procedures of the three kinds of MFs. The results of the analysis of the scattering (mean size of single polydisperse magnetic particles, fractal dimensions of the aggregates) are consistent with the data of transmission electron microscopy (TEM).

  10. Improved DNA electrophoresis in conditions favoring polyborates and lewis acid complexation.

    Directory of Open Access Journals (Sweden)

    Hari Singhal

    2010-06-01

    Full Text Available Spatial compression among the longer DNA fragments occurs during DNA electrophoresis in agarose and non-agarose gels when using certain ions in the conductive buffer, impairing the range of fragment sizes resolved well in a single gel. Substitutions using various polyhydroxyl anions supported the underlying phenomenon as the complexation of Lewis acids to DNA. We saw significant improvements using conditions (lithium borate 10 mM cations, pH 6.5 favoring the formation of borate polyanions and having lower conductance and Joule heating, delayed electrolyte exhaustion, faster electrophoretic run-speed, and sharper separation of DNA bands from 100 bp to 12 kb in a single run.

  11. 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 ...

  12. 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.

  13. In-house Manual Construction of High-Density and High-Quality Tissue Microarrays by Using Homemade Recipient Agarose-Paraffin Blocks.

    Science.gov (United States)

    Kim, Kyu Ho; Choi, Suk Jin; Choi, Yeon Il; Kim, Lucia; Park, In Suh; Han, Jee Young; Kim, Joon Mee; Chu, Young Chae

    2013-06-01

    Self-made tissue punches can be effectively used to punch holes in blank recipient paraffin blocks and extract tissue cores from the donor paraffin blocks for the low-cost construction of tissue microarrays (TMAs). However, variable degrees of section distortion and loss of the tissue cores can occurs during cutting of the TMAs, posing technical problems for in-house manual construction of high-density TMAs. We aimed to update the method for in-house manual TMA construction to improve the quality of high-density TMAs. Blocks of agarose gel were subjected to the standard tissue processing and embedding procedure to prepare recipient agarose-paraffin blocks. The self-made tissue punches and recipient agarose-paraffin blocks were used to construct TMAs, which were completely melted and re-embedded in paraffin to make finished TMA blocks. The donor tissue cores were completely integrated into the surrounding paraffin of the recipient blocks. This method enabled us to construct high-density TMAs with significantly less section distortion or loss of tissue cores during microtomy. Simple and inexpensive construction of high-density and high-quality TMAs can be warranted by using paraffinized agarose gels as recipient blocks.

  14. Crosslinked agarose encapsulated sorbents resistant to steam sterilization. Preparation and mechanical properties

    NARCIS (Netherlands)

    de Koning, H. W.; Chamuleau, R. A.; Bantjes, A.

    1984-01-01

    The application of agarose in hemoperfusion is hampered by the lack of a suitable sterilization method. A technique has been developed for the crosslinking of agarose encapsulated sorbents by the reaction with 1,3-dichloro-2-propanol (DCP) under strong alkaline conditions. A twofold molar excess of

  15. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    Science.gov (United States)

    Salehi, Nasrin; Peng, Ching-An

    2016-07-08

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

  16. Growth in Agarose of Human Cells Infected with Cytomegalovirus

    Science.gov (United States)

    Lang, David J.; Montagnier, Luc; Latarjet, Raymond

    1974-01-01

    After infection by human cytomegalovirus (CMV), human diploid fibroblasts could grow in agarose medium for several generations. Clones of infected cells grew for weeks, although in every case they ultimately underwent lysis owing to the cytopathic effect of the virus. Virus was inoculated at high dilution and after UV irradiation in an effort to derive cells infected with noninfectious defective particles still capable of inducing cell stimulation. Dilute or irradiated virus occasionally yielded large colonies of replicating cells, although permanent transformation was not observed. One clone derived from UV-CMV-infected cells was passaged four times before undergoing lysis. During these passages the cells exhibited alterations in morphology and orientation. Images PMID:4367907

  17. Thermoresponsive chitosan-agarose hydrogel for skin regeneration.

    Science.gov (United States)

    Miguel, Sónia P; Ribeiro, Maximiano P; Brancal, Hugo; Coutinho, Paula; Correia, Ilídio J

    2014-10-13

    Healing enhancement and pain control are critical issues on wound management. So far, different wound dressings have been developed. Among them, hydrogels are the most applied. Herein, a thermoresponsive hydrogel was produced using chitosan (deacetylation degree 95%) and agarose. Hydrogel bactericidal activity, biocompatibility, morphology, porosity and wettability were characterized by confocal microscopy, MTS assay and SEM. The performance of the hydrogel in the wound healing process was evaluated through in vivo assays, during 21 days. The attained results revealed that hydrogel has a pore size (90-400 μm) compatible with cellular internalization and proliferation. A bactericidal activity was observed for hydrogels containing more than 188 μg/mL of chitosan. The improved healing and the lack of a reactive or a granulomatous inflammatory reaction in skin lesions treated with hydrogel demonstrate its suitability to be used in a near future as a wound dressing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Molecular basis of an agarose metabolic pathway acquired by a human intestinal symbiont.

    Science.gov (United States)

    Pluvinage, Benjamin; Grondin, Julie M; Amundsen, Carolyn; Klassen, Leeann; Moote, Paul E; Xiao, Yao; Thomas, Dallas; Pudlo, Nicholas A; Anele, Anuoluwapo; Martens, Eric C; Inglis, G Douglas; Uwiera, Richard E R; Boraston, Alisdair B; Abbott, D Wade

    2018-03-13

    In red algae, the most abundant principal cell wall polysaccharides are mixed galactan agars, of which agarose is a common component. While bioconversion of agarose is predominantly catalyzed by bacteria that live in the oceans, agarases have been discovered in microorganisms that inhabit diverse terrestrial ecosystems, including human intestines. Here we comprehensively define the structure-function relationship of the agarolytic pathway from the human intestinal bacterium Bacteroides uniformis (Bu) NP1. Using recombinant agarases from Bu NP1 to completely depolymerize agarose, we demonstrate that a non-agarolytic Bu strain can grow on GAL released from agarose. This relationship underscores that rare nutrient utilization by intestinal bacteria is facilitated by the acquisition of highly specific enzymes that unlock inaccessible carbohydrate resources contained within unusual polysaccharides. Intriguingly, the agarolytic pathway is differentially distributed throughout geographically distinct human microbiomes, reflecting a complex historical context for agarose consumption by human beings.

  19. Preparation of gold nanoparticles-agarose gel composite and its application in SERS detection.

    Science.gov (United States)

    Ma, Xiaoyuan; Xia, Yu; Ni, Lili; Song, Liangjing; Wang, Zhouping

    2014-01-01

    Agarose gel/gold nanoparticles hybrid was prepared by adding gold nanoparticles to preformed agarose gel. Nanocomposite structures and properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV-Vis-NIR absorption spectroscopy. Based on the swelling-contraction characteristics of agarose gel and the adjustable localized surface plasmon resonance (LSPR) of the gold nanoparticles, the nanocomposites were used as surface enhanced Raman scattering (SERS) substrate to detect the Raman signal molecules (NBA, MBA, 1NAT). Results revealed that the porous structure of the agarose gel provided a good carrier for the enrichment of the gold nanoparticles. The gold nanoparticles dynamic hot-spot effect arising from the agarose gel contraction loss of water in the air greatly enhanced the Raman signal. Furthermore, the gel could be cleaned with washing solution and recycling could be achieved for Raman detection. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. 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.

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

  3. Distributed vasculogenesis from modular agarose-hydroxyapatite-fibrinogen microbeads.

    Science.gov (United States)

    Rioja, Ana Y; Daley, Ethan L H; Habif, Julia C; Putnam, Andrew J; Stegemann, Jan P

    2017-06-01

    Critical limb ischemia impairs circulation to the extremities, causing pain, disrupted wound healing, and potential tissue necrosis. Therapeutic angiogenesis seeks to repair the damaged microvasculature directly to restore blood flow. In this study, we developed modular, micro-scale constructs designed to possess robust handling qualities, allow in vitro pre-culture, and promote microvasculature formation. The microbead matrix consisted of an agarose (AG) base to prevent aggregation, combined with cell-adhesive components of fibrinogen (FGN) and/or hydroxyapatite (HA). Microbeads encapsulating a co-culture of human umbilical vein endothelial cells (HUVEC) and fibroblasts were prepared and characterized. Microbeads were generally 80-100µm in diameter, and the size increased with the addition of FGN and HA. Addition of HA increased the yield of microbeads, as well as the homogeneity of distribution of FGN within the matrix. Cell viability was high in all microbead types. When cell-seeded microbeads were embedded in fibrin hydrogels, HUVEC sprouting and inosculation between neighboring microbeads were observed over seven days. Pre-culture of microbeads for an additional seven days prior to embedding in fibrin resulted in significantly greater HUVEC network length in AG+HA+FGN microbeads, as compared to AG, AG+HA or AG+FGN microbeads. Importantly, composite microbeads resulted in more even and widespread endothelial network formation, relative to control microbeads consisting of pure fibrin. These results demonstrate that AG+HA+FGN microbeads support HUVEC sprouting both within and between adjacent microbeads, and can promote distributed vascularization of an external matrix. Such modular microtissues may have utility in treating ischemic tissue by rapidly re-establishing a microvascular network. Critical limb ischemia (CLI) is a chronic disease that can lead to tissue necrosis, amputation, and death. Cell-based therapies are being explored to restore blood flow and

  4. High lane density slab-gel electrophoresis using micromachined instrumentation.

    Science.gov (United States)

    Papautsky, I; Mohanty, S; Weiss, R; Frazier, A B

    2001-10-01

    In this paper, micromachined pipette arrays (MPAs) and microcombs were studied as a means of enabling high lane density gel electrophoresis. The MPA provide a miniaturized format to interface sub-microliter volumes of samples between macroscale sample preparation formats and microscale biochemical analysis systems. The microcombs provide a means of creating sample loading wells in the gel material on the same center-to-center spacing as the MPAs. Together, the two micromachined instruments provide an alternative to current combs and pipetting technologies used for creating sample loading wells and sample delivery in gel electrophoresis systems. Using three designs for the microcomb-MPA pair, center-to-center spacings of 1.0 mm, 500 microm, and 250 microm are studied. The results demonstrate an approximate 10-fold increase in lane density and a 10-fold reduction in sample size from 5 microL to 500 pL. As a result, the number of theoretical plates has increased 2.5-fold, while system resolution has increased 1.5-fold over the conventional agarose gel systems. An examination of changes in resolution across the width of individual separation lanes in both systems revealed dependence in the case of the conventional gels and no dependence for the gels loaded with the micromachined instrumentation.

  5. Comparative Two-Dimensional Fluorescence Gel Electrophoresis.

    Science.gov (United States)

    Ackermann, Doreen; König, Simone

    2018-01-01

    Two-dimensional comparative fluorescence gel electrophoresis (CoFGE) uses an internal standard to increase the reproducibility of coordinate assignment for protein spots visualized on 2D polyacrylamide gels. This is particularly important for samples, which need to be compared without the availability of replicates and thus cannot be studied using differential gel electrophoresis (DIGE). CoFGE corrects for gel-to-gel variability by co-running with the sample proteome a standardized marker grid of 80-100 nodes, which is formed by a set of purified proteins. Differentiation of reference and analyte is possible by the use of two fluorescent dyes. Variations in the y-dimension (molecular weight) are corrected by the marker grid. For the optional control of the x-dimension (pI), azo dyes can be used. Experiments are possible in both vertical and horizontal (h) electrophoresis devices, but hCoFGE is much easier to perform. For data analysis, commercial software capable of warping can be adapted.

  6. Proteoglycon synthesis by articular chondrocytes in agarose culture

    International Nuclear Information System (INIS)

    Sweet, M.B.E.; Grisillo, A.; Coehlo, A.; Schnitzler, C.M.

    1987-01-01

    Articular chondrocytes were isolated from knee joints of full-term bovine foetuses and grown in long-term agarose cultures. At intervals, cultures were labelled with 35 S-[sulphate] or D[6- 3 H] glucosamine. Newly synthesized proteoglycans were extracted with 4 M guanidine HCl and purified by isopycnic density gradient centrifugation or on DEAE cellulose in the presence of 8 M urea. Characterization of the proteoglycans revealed them to be identical in size to those present in the tissue and to be similarly capable of aggregation with hyaluronate. Newly synthesized chondroitin sulphate chains were identical in size, but newly synthesized keratan sulphate chains were somewhat larger than those present in the tissue. The newly synthesized proteoglycans were shown to contain the same range of O-linked oligosaccharides identified in proteoglycans of the Swarm rat chondrosarcoma. Cartilage-specific proteoglycan continued to be synthesized by the chondrocytes for up to 60 days; however, with time, proportionately more of a small non-aggregating proteoglycan appeared

  7. 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.

  8. RBC electrophoresis with discontinuous power supply - a newly established hemoglobin release test.

    Science.gov (United States)

    Su, Yan; Shao, Guo; Gao, Lijun; Zhou, Lishe; Qin, Liangyi; Qin, Wenbin

    2009-09-01

    In this paper, we aimed to introduce a newly established red blood cells (RBCs) electrophoresis method hemoglobin release test (HRT) and tried to determine its significance. Human blood samples from beta-thalassemia patients and healthy controls were analyzed with HRT, which was carried out on starch-agarose mixed gel. First, the whole blood samples were electrophoresed for 2 h, then paused for 15 min and ran for 15 min by turns. This "pause-run-pause" experiment was performed for several turns and the total electrophoresis time lasted for about 6 h. The results showed that some other hemoglobin (Hb) components were released from the origin of each sample during the HRT, and the samples from beta-thalassemia patients released more Hb than the healthy controls. This finding demonstrates that Hb may exist differently associated in RBCs, and it may have an important theoretical and clinical significance in Hb and RBC research.

  9. Protein blotting with direct blotting electrophoresis.

    Science.gov (United States)

    Beck, S

    1988-05-01

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

  10. In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes

    OpenAIRE

    Brugère, Jean-François; Cornillot, Emmanuel; Méténier, Guy; Vivarès, Christian P.

    2000-01-01

    International audience; A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [α-32 P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view ...

  11. Electrophoresis and electro-affinity transfer with specific antibodies to alpha-fetoprotein for detection of circulating immune complexes of alpha-fetoprotein.

    OpenAIRE

    Taketa, Kazuhisa; Ichikawa, Eriko; Taga, Hiroko; Hirai, Hidematsu

    1984-01-01

    A combination of agarose gel electrophoresis and a newly developed technique of electro-affinity transfer was applied to the detection of circulating immune complexes of human alpha-fetoprotein (AFP) and anti-AFP. After electrophoretic transfer to nitrocellulose membrane, to which affinity-purified polyclonal horse antibodies to human AFP were bound, the membranes were treated with or without rabbit immunoglobulins to human AFP, followed by overlaying with horseradish peroxidase-labeled goat ...

  12. Electrophoretic study of whey proteins in Holstein cows with clinical and subclinical mastitis by Agarose gel procedure

    Directory of Open Access Journals (Sweden)

    A Davasaz Tabrizi

    2009-02-01

    Full Text Available Mastitis is one the most important economic diseases in dairy cattle industry, which causes reduction in milk production, treatment expenses, reduction in herd genetic progress and fall in quality of milk. The aim of this study was to examine the milk proteins of Holstein dairy cows with different grades of clinical and subclinical mastitis. During the sampling period, none of the cows were in late pregnancy or at early lactation and also had no parasitemia and any other inflammatory diseases.  Clinical and laboratory examinations which were carried out completely revealed the cows were all healthy. They were fed on corn silage, concentrate and alfalfa. In this study, the cows were divided into five groups, each group with 25 cases. For this purpose, milk samples were collected from 125 dairy cattle of two large dairy farms in Tabriz. All the cows were in the lactation period and they were milked three times a day. The groups consist of the control group with negative California mastitis test and negative culture, 2+ subclinical groups, 3+ subclinical group, sub acute clinical group and acute clinical group. The results of the whey electrophoresis using Agarose gel procedure indicated significant difference in albumin levels in all groups except the 2+ subclinical group compared with the control group (p

  13. Characterization of β -Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose.

    Science.gov (United States)

    Baraldo Junior, Anderson; Borges, Diogo G; Tardioli, Paulo W; Farinas, Cristiane S

    2014-01-01

    β -Glucosidase (BGL) is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger cultivated under solid-state fermentation (SSF) was partially purified and fully characterized in terms of optimum pH, temperature, and thermostability. The single-step purification using MANAE-agarose in a chromatographic column yielded an enzyme solution with specific activity (17.1 IU/mg protein) adequate for the characterization procedures. Electrophoresis SDS-PAGE and size-exclusion chromatography analysis resulted in an estimated molecular mass of 60 kDa. Higher enzyme activities were found in the range between 40 and 65°C and between pH 4 and 5.5, indicating an interesting characteristic for application in the hydrolysis of lignocellulosic biomass for biofuels production. Thermostability studies of purified BGL resulted in half-lives at 37°C of 56.3 h and at 50°C of 5.4 h. These results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

  14. Films of Agarose Enable Rapid Formation of Giant Liposomes in Solutions of Physiologic Ionic Strength

    OpenAIRE

    Horger, Kim S.; Estes, Daniel J.; Capone, Ricardo; Mayer, Michael

    2009-01-01

    This paper describes a method to form giant liposomes in solutions of physiologic ionic strength, such as phosphate buffered saline (PBS) or 150 mM KCl. Formation of these cell-sized liposomes proceeded from hybrid films of partially dried agarose and lipids. Hydrating the films of agarose and lipids in aqueous salt solutions resulted in swelling and partial dissolution of the hybrid films and in concomitant rapid formation of giant liposomes in high yield. This method did not require the pre...

  15. In vitro ovarian cancer model based on three-dimensional agarose hydrogel

    Directory of Open Access Journals (Sweden)

    Guojie Xu

    2014-01-01

    Full Text Available To establish a typical tumor model of ovarian cancer which may be more representative and reliable than traditional monolayer culture and pellet, agarose was used as cell vehicle to engineering tumor. Selection of agarose is based on its successful application in tissue engineering with both amenable mechanical and biological properties. In this study, ovarian cancer cell line SKOV3 was encapsulated in agarose hydrogel with cell aggregates and two-dimensional culture as controls. In vitro cell proliferation was assessed by MTT and cell viability was examined at time points of 2, 4, and 6 days. The expression of tumor malignancy markers including matrix metalloproteinase 2, matrix metalloproteinase 9, hypoxia-inducible factor-1α, and vascular endothelial growth factor–A was assessed by real-time polymerase chain reaction. The results showed that cells proliferated more rapidly in three-dimensional agarose culture than controls. Furthermore, upregulation of matrix metalloproteinase 9 and matrix metalloproteinase 2 activity and increased expression of vascular endothelial growth factor–A and hypoxia-inducible factor-1α were shown in agarose-engineered tumors. All the evidences demonstrated that agarose may provide a more favorable environment for cancer cell growth, mimicking the in vivo environment for tumor generation. The novel in vitro tumor model may be useful for the further investigation of anticancer therapeutics.

  16. 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

  17. 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

  18. Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

    Science.gov (United States)

    Fiscus, R R; Leung, C P; Yuen, J P; Chan, H C

    2001-01-01

    Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines. Copyright 2001 Academic Press.

  19. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

    Energy Technology Data Exchange (ETDEWEB)

    Dumpala, Pradeep R., E-mail: pdumpala@rixd.org [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Parker, Thomas S.; Levine, Daniel M. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); Smith, Barry H. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); NewYork-Presbyterian Hospital, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021 (United States); Gazda, Lawrence S. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States)

    2016-08-05

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

  20. Agarose overlay selectively improves macrocolony formation and radiosensitivity assessment in primary fibroblasts.

    Science.gov (United States)

    Chandna, Sudhir; Dagur, Raghubendra Singh; Mathur, Ankit; Natarajan, Adayapalam Tyagarajan; Harms-Ringdahl, Mats; Haghdoost, Siamak

    2014-05-01

    Primary fibroblasts are not suitable for in vitro macrocolony assay due to their inability to form distinct colonies. Here we present a modification of agarose overlay that yielded extensive improvement in their colony formation and assessment of radiosensitivity. Macrocolony formation was assessed in primary human fibroblasts VH10 and HDFn with or without overlay using 0.5% agarose in growth medium at 24 h post-seeding. Malignant human cell lines (A549, U87) and transformed non-malignant fibroblasts (AA8 hamster, MRC5 human) were used for comparison. Agarose overlay caused significant improvement marked by early appearance (one week) of distinct colonies with high cell density and multifold higher plating efficiency than conventional macrocolony assay in VH10 and HDFn human fibroblasts. Compared to conventional assay or feeder cell supplementation, agarose overlay resulted in broader cell morphology due to improved adherence, and yielded more compact colonies. Gamma-radiation dose-response survival curves could be successfully generated for both fibroblast cell lines using this method, which yielded no such effects in the transformed/malignant cell lines tested. This easy and inexpensive 'agarose overlay technique' significantly and selectively improves the fibroblast plating efficiency, thus considerably reducing time and effort to greatly benefit the survival studies on primary fibroblasts.

  1. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

    International Nuclear Information System (INIS)

    Dumpala, Pradeep R.; Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A.; Parker, Thomas S.; Levine, Daniel M.; Smith, Barry H.; Gazda, Lawrence S.

    2016-01-01

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

  2. Quantitative determination of glycine in aqueous solution using glutamate dehydrogenase-immobilized glyoxal agarose beads.

    Science.gov (United States)

    Keskin, Semra Yilmazer; Keskin, Can Serkan

    2014-01-01

    In this study, an enzymatic procedure for the determination of glycine (Gly) was developed by using a column containing immobilized glutamate dehydrogenase (GDH) on glyoxal agarose beads. Ammonia is produced from the enzymatic reactions between Gly and GDH with NAD(+) in phosphate buffer medium. The indophenol blue method was used for ammonia detection based on the spectrophotometric measurements of blue-colored product absorbing at 640 nm. The calibration graph is linear in the range of 0.1-10 mM of Gly concentrations. The effect of pH, temperature, and time interval was studied to find column stability, and also the interference effects of other amino acids was investigated. The interaction between GDH and glyoxal agarose beads was analyzed by Fourier transform infrared (FTIR) spectroscopy. The morphology of the immobilized and non-immobilized agarose beads were characterized by atomic force microscopy (AFM).

  3. Fenugreek hydrogel-agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection.

    Science.gov (United States)

    Kestwal, Rakesh Mohan; Bagal-Kestwal, Dipali; Chiang, Been-Huang

    2015-07-30

    A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel-agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10-20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel-agarose-acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients.

    Science.gov (United States)

    Khosravi, Azar Dokht; Vatani, Shideh; Feizabadi, Mohammad Mehdi; Abasi Montazeri, Effat; Jolodar, Abbas

    2014-05-01

    Mycobacterium tuberculosis genotyping can effectively improve tuberculosis (TB) control programs by controlling disease transmission. Pulsed field gel electrophoresis (PFGE) is a particularly powerful tool for determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. The aim of present study was to investigate the genetic diversity of M. tuberculosis strains in Khuzestan province by the PFGE technique. In total, 80 M. tuberculosis positive cultures were obtained from tuberculosis patients. PFGE was performed on 60 PCR-confirmed isolates by using DraI and XbaI restriction enzymes according to standard protocols. Plugs containing digested DNA were then loaded on agarose gels and run using contour-clamped homogenous electric fields. Fifty distinct DNA banding patterns were obtained by digestion of DNA with DraI and 38 DNA banding patterns by digestion with XbaI restriction enzymes. The patterns comprised of 17 different clusters in which cluster I was the major one, containing six strains. Three clusters contained three strains each and the 13 remaining clusters comprised of two strains each. Digestion with DraI yielded 15-20 DNA fragments with 50-485 kb size, while digestion by XbaI produced DNA fragments with a size smaller than 50-242 kb. Despite the ability of PFGE for study of genetic diversity of many mycobacterial species and it being considered as a robust and useful tool, in this study we only found a 15% epidemiological relationship amongst the isolates. Thus, for higher discrimination of genotypic clusters among M. tuberculosis clinical isolates, the application of more sophisticated complementary techniques is required.

  5. Primary Separation: 2-D Electrophoresis

    NARCIS (Netherlands)

    Pedreschi Plasencia, R.P.

    2013-01-01

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

  6. Measurement of Ferric Ion Diffusion Coefficient in Fricke-Infused Agarose Gel From MR Image Intensity Changes

    National Research Council Canada - National Science Library

    Tseng, Yin-Jiun

    2001-01-01

    .... Our results showed that for a Fricke-agarose gel contained 1mM ammonium ferrous sulfate, 1% agarose, 1mM sodium chloride and 50mM sulfuric acid, its ferric ion diffusion coefficient is 1.31x10(-2)cm(2)h(-1...

  7. Radiation-induced DNA damage and repair in radiosensitive and radioresistant human tumour cells measured by field inversion gel electrophoresis

    International Nuclear Information System (INIS)

    Smeets, M.F.M.A.; Mooren, E.H.M.; Begg, A.C.

    1993-01-01

    Radiation-induced DNA damage induction and repair was measured in two human squamous carcinoma cell lines with differing radiosensitivities. Experiments were carried out with field inversion gel electrophoresis (FIGE), adapted to measure DNA double strand break (DSB) induction and repair in unlabelled cells. The sensitivity of the method was increased by introducing a hybridization membrane into the agarose gel. Damaged DNA accumulated on one spot on the membrane resulting in high local concentrations. This DNA was quantified using radioactively-labelled total human DNA as a probe. Radiosensitivity differences at physiological temperatures could not be explained by differences in either induction or repair of DNA damage as measured by pulsed field gel electrophoresis. (author)

  8. Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1994-01-01

    Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10 B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

  9. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Dept. de Biofisica e Biometria. Lab. de Radio e Fotobiologia]. E-mail: jcmattos@uerj.br

    2008-12-15

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl{sub 2}) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl{sub 2} in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl{sub 2} was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  10. Rapid recovery of DNA from agarose gel slices by coupling electroelution with monolithic SPE.

    Science.gov (United States)

    Yu, Shengbing; Yang, Shuixian; Zhou, Ping; Zhou, Ke; Wang, Jing; Chen, Xiangdong

    2009-06-01

    An amino silica monolithic column prepared by in situ polymerization of tetraethoxysilane and N-(beta-aminoethyl)-gamma-aminopropyltriethoxysilane was firstly applied to recover DNA from agarose gel slices by coupling electroelution with monolithic SPE. DNA was electroeluted from the agarose gel slices onto the amino silica monolithic column. The DNA adsorbed on this monolithic column was then recovered using sodium phosphate solution at pH 10. The whole recovery procedure could be completed within 10 min because the use of amino silica monolithic column accelerated the DNA capture and facilitated the DNA release. Electroelution conditions, such as buffer pH, buffer concentration and applied voltage, were online optimized. The average yield for herring sperm DNA, pBR 322 DNA and lambda DNA recovered from 1.0% w/v agarose gel slices were 55+/-4, 50+/-6 and 42+/-7% (n=3), respectively. The polymerase chain reaction performance of pGM plasmid recovered from agarose gel slices demonstrated that the method could provide high-quality DNA for downstream processes. The combination of electroelution with monolithic SPE allows a rapid, simple and efficient DNA recovery method. This technique is especially useful for applications that need to purify small starting amounts of DNA.

  11. Effects of calcium salts of acidic monomers on mineral induction of phosphoprotein immobilized to agarose beads.

    Science.gov (United States)

    Ito, Shuichi; Iijima, Masahiro; Motai, Fumiko; Mizoguchi, Itaru; Saito, Takashi

    2012-10-01

    The aim of this study is to evaluate the mineralizing potential of acidic monomers and their calcium salts for mineralization, using an in vitro mineral induction model. Phosvitin (PV) was used as a model phosphoprotein in this study. PV was immobilized on agarose beads with divinyl sulfone. Five aliquots of agarose-immobilized PV, acidic monomers, and their calcium salts were incubated in mineralizing solution at various concentrations. The PV beads and acidic monomers were incubated at 37°C. Samples were taken at several time points during the incubation. Then, the agarose beads were analyzed for bound calcium by atomic absorption spectrometry. The mineral formed on the agarose beads was identified as an apatite by microarea X-ray diffraction. Additionally, the specimens were observed using scanning electron microscopy (SEM). Mineral induction time decreased with increasing solution saturation. 4-METCa salt [calcium salt of 4-methacryloxyethyl trimellitate (CMET)] significantly reduced the mineral induction time. Using these data, the interfacial tension for mineral induction of PV and CMET was determined to be 90.1 and 92.7 ergs/cm(2), respectively. The mineral induced in each specimen after incubation for 24 h was identified by its X-ray diffraction pattern as apatite. SEM observation showed that lath-shaped crystals were formed on the surfaces of the CMET. We conclude that CMET could play a role in dentin remineralization. Copyright © 2012 Wiley Periodicals, Inc.

  12. Direct Quantification of Solute Diffusivity in Agarose and Articular Cartilage Using Correlation Spectroscopy.

    Science.gov (United States)

    Shoga, Janty S; Graham, Brian T; Wang, Liyun; Price, Christopher

    2017-10-01

    Articular cartilage is an avascular tissue; diffusive transport is critical for its homeostasis. While numerous techniques have been used to quantify diffusivity within porous, hydrated tissues and tissue engineered constructs, these techniques have suffered from issues regarding invasiveness and spatial resolution. In the present study, we implemented and compared two separate correlation spectroscopy techniques, fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), for the direct, and minimally-invasive quantification of fluorescent solute diffusion in agarose and articular cartilage. Specifically, we quantified the diffusional properties of fluorescein and Alexa Fluor 488-conjugated dextrans (3k and 10k) in aqueous solutions, agarose gels of varying concentration (i.e. 1, 3, 5%), and in different zones of juvenile bovine articular cartilage explants (i.e. superficial, middle, and deep). In agarose, properties of solute diffusion obtained via FCS and RICS were inversely related to molecule size, gel concentration, and applied strain. In cartilage, the diffusional properties of solutes were similarly dependent upon solute size, cartilage zone, and compressive strain; findings that agree with work utilizing other quantification techniques. In conclusion, this study established the utility of FCS and RICS as simple and minimally invasive techniques for quantifying microscale solute diffusivity within agarose constructs and articular cartilage explants.

  13. Immunoperoxidase staining and radioimmunobinding of human tumor markers separated by direct tissue agarose isoelectric focusing

    International Nuclear Information System (INIS)

    Saravis, C.A.; Cunningham, C.G.; Marasco, P.V.; Cook, R.B.; Zamcheck, N.; FMC Corp., Rockland, ME

    1980-01-01

    The new technique of agarose isoelectric focusing is used to identify, quantitate, and characterize specific tumor markers. After fixation of the isoelectric focusing patterns these are reacted with specific anti-tumor marker antisera, then with second antibody either peroxidase conjugated or radiolabellad (radioiodine). (RB) [de

  14. Simultaneous DNA purification and fractionation in agarose gel on the micro-scale

    NARCIS (Netherlands)

    Gümüscü, B.; van den Berg, Albert; Eijkel, Jan C.T.

    2016-01-01

    We report a new and simple approach for preparative purification and fractionation of sub-10-kbp DNA molecules in a microfluidic device. Agarose gel with 1.2% concentration is used as the separation matrix. A 0.5-10 kbp DNA ladder is fractionated and separated from other ionic species in continuous

  15. Micropatterning of a stretchable conductive polymer using inkjet printing and agarose stamping

    DEFF Research Database (Denmark)

    Hansen, Thomas Steen; Hassager, Ole; Larsen, Niels Bent

    2007-01-01

    ,4-ethylenedioxythiophene) (PEDOT). The agarose stamping produced 50 μm wide conducting lines with high spatial fidelity. The deactivation agent was found to cause some degradation of the remaining conducting lines, as revealed by a stronger increase in resistance upon straining compared to the pristine polymer material...

  16. A colorimetric agarose gel for formaldehyde measurement based on nanotechnology involving Tollens reaction.

    Science.gov (United States)

    Zeng, Jing-bin; Fan, Shi-guang; Zhao, Cui-ying; Wang, Qian-ru; Zhou, Ting-yao; Chen, Xi; Yan, Zi-feng; Li, Yan-peng; Xing, Wei; Wang, Xu-dong

    2014-08-04

    Gold nanoparticles (Au NPs) coupled with Tollens reagent were used for measuring formaldehyde. Au@Ag core-shell NPs were formed along with distinct color changes from pink to deep yellow. This colorimetric system was further immobilized into an agarose gel, which was used for monitoring of gaseous formaldehyde.

  17. A simple immunoblotting method after separation of proteins in agarose gel

    DEFF Research Database (Denmark)

    Koch, C; Skjødt, K; Laursen, I

    1985-01-01

    A simple and sensitive method for immunoblotting of proteins after separation in agarose gels is described. It involves transfer of proteins onto nitrocellulose paper simply by diffusion through pressure, a transfer which only takes about 10 min. By this method we have demonstrated the existence ...

  18. Polyacrylamide gel electrophoresis of semiconductor quantum dots and their bioconjugates: materials characterization and physical insights from spectrofluorimetric detection.

    Science.gov (United States)

    Kim, Hyungki; Jeen, Tiffany; Tran, Michael V; Algar, W Russ

    2018-02-26

    Colloidal semiconductor quantum dot (QD) nanocrystals have ideal fluorescence properties for bioanalysis and bioimaging, but these materials must be functionalized with an inorganic shell, organic ligand or polymer coating, and conjugated with biomolecules to be useful in such applications. Several different analytical techniques are used to characterize QDs and their multiple layers of functionalization. Here, we revisit poly(acrylamide) gel electrophoresis (PAGE), which has been scarcely used for the characterization of QDs and their bioconjugates in deference to the routine use of agarose gel electrophoresis. We implemented PAGE in a novel "stubby" capillary format with spectrofluorimetric detection, the combination of which enabled more rapid and more detailed characterization of QDs than was possible with both poly(acrylamide) and agarose slab gels. Correlations between the peak photoluminescence (PL) emission wavelength and electropherogram peaks, especially when combined with Ferguson analysis, provided new and significant insight into the key factors that determine the electrophoretic mobility of QDs, and helped to resolve heterogeneity and sub-populations in ensembles of QDs. The method was useful for characterization of the inorganic core/shell nanocrystals, their organic ligand and polymer coatings, and their final bioconjugates, the latter of which were in the form of peptide and protein conjugates. With further development and optimization, we anticipate that capillary PAGE with spectrofluorimetric detection will become a valuable addition to the toolbox of characterization techniques suitable for QDs, their bioconjugates, and other nanoparticle materials as well.

  19. Determination of the Mutagenicity Potential of Supermint Herbal Medicine by Single Cell Gel Electrophoresis in Rat Hepatocytes

    Directory of Open Access Journals (Sweden)

    Zivar Amanpour

    2012-08-01

    Full Text Available Purpose: The increasing use of herbal drugs and their easy availability have necessitated the use of mutagenicity test to analyze their toxicity and safety. The aim of this study was to evaluate the genotoxicity of Supermint herbal medicine in DNA breakage of rat hepatocytes in comparison with sodium dichromate by single cell gel electrophoresis technique or comet assay. Methods: Hepatocytes were prepared from male wistar rats and were counted and kept in a bioreactor for 30 minutes. Then cells were exposed to the Supermint herbal medicine at doses of 125, 250 and 500 μl/ml. Buffer 4 (incubation buffer and sodium dichromate were used as negative and positive control for one hour respectively. Then cell suspension with low melting point agarose were put on precoated slides and covered with agarose gel. Then lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. The parameter under this analysis was the type of migration which was determined according to Kobayashi pattern. Results: With increased dose of Supermint herbal medicine the DNA damage was slightly increased (P<0001. Conlusion: In overall compared to the positive control significant differences is observed which convinced that the crude extract of Supermint in vitro did not have mutagenic effect.

  20. Ratcheted electrophoresis of Brownian particles

    Energy Technology Data Exchange (ETDEWEB)

    Kowalik, Mikołaj; Bishop, Kyle J. M., E-mail: kjmbishop@engr.psu.edu [Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802 (United States)

    2016-05-16

    The realization of nanoscale machines requires efficient methods by which to rectify unbiased perturbations to perform useful functions in the presence of significant thermal noise. The performance of such Brownian motors often depends sensitively on their operating conditions—in particular, on the relative rates of diffusive and deterministic motions. In this letter, we present a type of Brownian motor that uses contact charge electrophoresis of a colloidal particle within a ratcheted channel to achieve directed transport or perform useful work against an applied load. We analyze the stochastic dynamics of this model ratchet to show that it functions under any operating condition—even in the limit of strong thermal noise and in contrast to existing ratchets. The theoretical results presented here suggest that ratcheted electrophoresis could provide a basis for electrochemically powered, nanoscale machines capable of transport and actuation of nanoscale components.

  1. Detection of the irradiation treatment of foods using micro gel electrophoresis of DNA

    International Nuclear Information System (INIS)

    Delincee, H.

    1993-01-01

    Ionizing radiation has profound effects on nucleic acids, reflected by e.g. base damage and strandbreaks. These effects of radiation contribute to the aim of food irradiation: undesirable microorganisms are inactivated, insect infestation eliminated, sprouting inhibited or ripening delayed. It is, therefore, conceivable that methods of detection of a radiation treatment could be based on changes in DNA, either in microbes or insects or in the food itself. A promising method of detecting DNA fragments in irradiated food is the microelectrophoresis of single cells. Advantages of the method are its simplicity and its speed, the electrophoretic separation only requiring 2.5-5 minutes. The principle is based on migration of DNA in an agarose gel exposed to an electric field. Single cells or nuclei are embedded in the agarose and after lysis intact DNA will practically not move upon electrophoresis, due to the gel strains, whereas if DNA fragmentation has occurred, the DNA is able to migrate and ''comets'' following the cells or nuclei become visible after staining. This paper describes our experience obtained by analyzing chicken, beef and pork meat, shrimps and mushrooms. The method therefore has its limitations, although good results both from frozen and fresh meats, fresh fish, onions and potatoes have been reported. Also strawberries have been identified using this technique. Further experience may strengthen the evidence that this simple and cheap technique may have its place among the battery of tests to detect the radiation treatment of foods. (orig./vhe)

  2. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  3. Two staining methods for selectively detecting isomaltase and maltase activity in electrophoresis gels.

    Science.gov (United States)

    Finlayson, S D; Moore, P A; Johnston, J R; Berry, D R

    1990-05-01

    Two methods for specifically detecting maltase, alpha-glucosidase, or isomaltase activity in electrophoresis gels are described. Both systems couple the formation of glucose by enzyme action on maltose or isomaltose to the generation of a colored product. System A uses an agarose overlay which contains substrate, glucose oxidase, peroxidase, 2,4-dichlorophenol, and 4-L-amino-phenazone. A purple color is produced at the site of enzyme activity. No hazardous chemicals are used at any stage. The stain is simple, rapid, sensitive, and inexpensive and does not interfere with subsequent protein staining. However, the stain is not permanent. System B was developed to give a permanent stain. The gel is overlaid with agarose containing substrate, glucose oxidase, phenazine methosulfate, and nitroblue tetrazolium. Glucose production results in the nitroblue tetrazolium being oxidized to an insoluble formazan with a dark blue color. This stain is also sensitive, rapid, and inexpensive but does use hazardous chemicals and if overstaining occurs this can interfere with subsequent protein staining. Neither system inactivates the localized enzymes which can be recovered from the gel if desired.

  4. 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

  5. DNA-electrophoresis of single cells - a method to screen for irradiated foodstuffs

    International Nuclear Information System (INIS)

    Leffke, A.; Helle, N.; Boegl, K.W.; Schreiber, G.A.

    1993-01-01

    Microelectrophoresis of single cells can be used to detect γ-irradiation over a wide dose range and for a variety of products. It is a simple and rapid test for DNA damages and can be used for screening. The method was tested on cell suspensions of bone marrow and muscle cells from frozen chicken legs, chicken heart, turkey liver, beef and pork irradiated with doses up to 3 kGy. Cell suspensions were prepared by incubation of tissues in EDTA-SDS-buffer at pH 8. Single cell electrophoresis was performed in 0.75% agarose gel. DNA was visualised by silver staining. In unirradiated samples no or only a small amount of DNA penetrated the cell membranes. Cells of irradiated samples appeared like a ''comet'' due to to migration of DNA-fragments out of cell. (orig.)

  6. Two-dimensional gel electrophoresis: glass tube-based IEF followed by SDS-PAGE.

    Science.gov (United States)

    Matsumoto, Hiroyuki; Haniu, Hisao; Kurien, Biji T; Komori, Naoka

    2012-01-01

    The genome information combined with data derived from modern mass spectrometry enables us to determine the identity of a protein once it is isolated from a complex mixture. Two-dimensional gel electrophoresis established more than three decades ago serves as a powerful protocol to isolate many proteins at once for such protein analysis. In the first two decades, the original procedure to use a glass tube-based isoelectric focusing (IEF) had been commonly used. Since an IEF in glass tubes is rather difficult to maneuver, a new method to use an IEF on a thin agarose slab backed by a plastic film (IPG Dry Strip) has been invented and is now widely used. In this chapter, we describe the original protocol that uses a glass tube-based IEF because, the capacity of protein loading and resolving power of this type of classic two-dimensional gel is still indispensible.

  7. Physicochemical, morphological and therapeutic evaluation of agarose hydrogel particles as a reservoir for basic fibroblast growth factor.

    Science.gov (United States)

    Moribe, Kunikazu; Nomizu, Natsuko; Izukura, Shunsuke; Yamamoto, Keiji; Tozuka, Yuichi; Sakurai, Manabu; Ishida, Atsushi; Nishida, Hirofumi; Miyazaki, Masaru

    2008-01-01

    Micron-sized agarose hydrogel particles were prepared using an emulsification/gelation method as a controlled release reservoir for basic fibroblast growth factor (bFGF). Mean particle size of agarose hydrogel particles decreased with an increase in stirring speed and also with an increasing temperature of the oil phase, as measured before cooling. Morphologies of agarose particles before and after dispersing into water were investigated by scanning electron microscopy (SEM) and cryogenic SEM, respectively. Freeze-dried agarose particles were spherical with rough surface. Porous polymer matrix structure was observed in the hydrogel particles by cryo-SEM. More than 99% of bFGF was encapsulated and the release from the agarose hydrogel particles was less than 3% during the incubation in phosphate buffered saline. bFGF molecules were not only adsorbed on the particle surface but also permeated and retained within the matrix. The therapeutic efficacy of bFGF retained in agarose hydrogel particles was significantly higher than that dissolved in saline. Agarose hydrogel particle seems to be a potential candidate for a bFGF reservoir.

  8. Structural aspects of magnetic fluid stabilization in aqueous agarose solutions

    Energy Technology Data Exchange (ETDEWEB)

    Nagornyi, A.V. [Joint Institute for Nuclear Research, Dubna (Russian Federation); Taras Shevchenko National University of Kyiv, Kyiv (Ukraine); Petrenko, V.I., E-mail: vip@nf.jinr.ru [Joint Institute for Nuclear Research, Dubna (Russian Federation); Taras Shevchenko National University of Kyiv, Kyiv (Ukraine); Avdeev, M.V. [Joint Institute for Nuclear Research, Dubna (Russian Federation); Yelenich, O.V.; Solopan, S.O.; Belous, A.G. [V.I.Vernadsky Institute of General and Inorganic Chemistry of the Ukrainian NAS, Kyiv (Ukraine); Gruzinov, A.Yu. [National Research Centre “Kurchatov Institute”, Moscow (Russian Federation); Ivankov, O.I. [Joint Institute for Nuclear Research, Dubna (Russian Federation); Institute for Safety Problems of Nuclear Power Plants of the Ukrainian NAS, Kyiv (Ukraine); Bulavin, L.A. [Taras Shevchenko National University of Kyiv, Kyiv (Ukraine); Institute for Safety Problems of Nuclear Power Plants of the Ukrainian NAS, Kyiv (Ukraine)

    2017-06-01

    Structure characterization of magnetic fluids (MFs) synthesized by three different methods in aqueous solutions of agarose was done by means of small-angle neutron (SANS) and synchrotron X-ray scattering (SAXS). The differences in the complex aggregation observed in the studied magnetic fluids were related to different stabilizing procedures of the three kinds of MFs. The results of the analysis of the scattering (mean size of single polydisperse magnetic particles, fractal dimensions of the aggregates) are consistent with the data of transmission electron microscopy (TEM). - Highlights: • MFs synthesized by three different methods in agarose solution were studied. • all MFs are agglomerated colloidal systems whose structures are nevertheless stable in time. • differences in the complex aggregation were observed in the studied magnetic fluids. • results of the SAXS and SANS analysis are consistent with TEM data.

  9. Preparation and structural characterization of O-acetyl agarose with low degree of substitution

    Directory of Open Access Journals (Sweden)

    Rosangela B. Garcia

    2000-09-01

    Full Text Available Among the biodegradable polymers, the polysaccharides have been found to be promising carriers for bioactive molecules. From a general standpoint, they present several reactive groups, such as hydroxyl, carboxyl and amine, that can be modified in a number of ways, giving rise to suitable devices for controlled release. In this paper, agarose was submitted to O-acetylation reactions under heterogeneous conditions, using acetic anhydride and pyridine, aiming to observe the effect of acetyl groups on the agarose properties. The products were characterized by Infrared and ¹H NMR spectroscopies. In the range of average acetylation degrees (DA 0.07-0.48, the polymers presented partial solubility in boiling water and in common organic solvents. The ¹H NMR spectra presented evidences of non-homogeneous acetyl group distribution along the chains, as concluded from the solubility of only one of the fractions with DA<0.09, in boiling water .

  10. Mechanics and transport phenomena in agarose-based hydrogels studied by compression-relaxation tests.

    Science.gov (United States)

    Caccavo, Diego; Cascone, Sara; Poto, Serena; Lamberti, Gaetano; Barba, Anna Angela

    2017-07-01

    Hydrogels are widespread materials, used in several frontier fields, due to their peculiar behavior: they couple solvent mass transport to system mechanics, exhibiting viscoelastic and poroelastic characteristics. The full understanding of this behavior is crucial to correctly design such complex systems. In this study agarose gels has been investigated through experimental stress-relaxation tests and with the aid of a 3D poroviscoelastic model. At the investigated experimental conditions, the agarose gels samples show a prevalent viscoelastic behavior, revealing limited water transport and an increase of the stiffness as well as of the relaxation time along with the polymer concentration. The model parameters, derived from the fitting of some experimental data, have been generalized and used to purely predict the behavior of another set of gels. The stress-relaxation tests coupled with mathematical modeling demonstrated to be a powerful tool to study hydrogels' behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Capillary electrophoresis theory and practice

    CERN Document Server

    Grossman, Paul D

    1992-01-01

    This book is designed to be a practical guide, used by wide audience, including those new to CE, those more experienced, routine users, those interested in technology development, and those involved with applications research. References have been emphasized to allow the reader to explore the detailed specifics and theoretical foundations.This book draws together the rapidly evolving, diverse, and multidisciplinary subject of capillary electrophoresis (CE). It is designed as a practical guide to be used by a wide audience, including those new to CE as well as more experienced users. T

  12. The Influence of Conditioning Agent on Phosphate Diffusion Coefficient through Polyacrylamide and Agarose Gel

    Directory of Open Access Journals (Sweden)

    Layta Dinira

    2013-03-01

    Full Text Available Excess phosphate in natural water can cause algae grow rapidly, to the extent causing many fish deaths that led to the extinction of certain species. Therefore, an analysis or periodic observations of phosphate levels in the water is needed. The commonly used method is diffusive gradient in thin films (DGT technique. The DGT technique is based on the ability of analyte to diffuse through a gel, which have a value named diffusion coefficient. This research was conducted in order to study the effect of different storage solution to the phosphate diffusion coefficient through polyacrylamide and agarose gels. Initial research performed with making the polyacrylamide and agarose gels. To observe the effect of different storage solutions, the gels partly stored in distilled water gel while the others are stored in a NaCl solution of 0.01 M. Phosphate diffusion coefficient was determined using Fick's Law after analyze the phosphate concentration using UV-Visible spectrophotometer. The results showed that phosphate diffusion coefficient was highest when polyacrylamide and agarose gels stored in NaCl solution of 0.01 M.

  13. Cryopreservation of very low numbers of spermatozoa from male patients undergoing infertility treatment using agarose capsules.

    Science.gov (United States)

    Hatakeyama, Shota; Tokuoka, Susumu; Abe, Hiroyuki; Araki, Yasuyuki; Araki, Yasuhisa

    2017-07-01

    This study tried to cryopreserve low numbers of spermatozoa from men undergoing infertility treatments by inserting into agarose capsules. The capsules were transferred into a drop of cryoprotectant solution and injected 3-4 motile spermatozoa that were selected by the swim-up method by conventional intracytoplasmic sperm injection. These capsules were put on a Cryotop ® and frozen in liquid nitrogen vapor, and then submerged into liquid nitrogen and subsequently thawed and recovered. The motile spermatozoa in the capsules were counted. Eventually, we cryopreserved 2142 motile spermatozoa in 702 agarose capsules from 26 male patients and 1356 (63%) spermatozoa maintained their motility after thawing. The spermatozoa motility rates after thawing (MRAT) ranged from 20.0% (5/25) to 95.1% (58/61) among patients. The median MRAT was 68.3% (interquartile range 46.1-75.7). The total number of motile spermatozoa collected by swim-up method strongly correlated with MRAT (r = 0.746). It was possible to cryopreserve spermatozoa from male patients undergoing infertility treatment using agarose capsules. However, there were wide differences in MRAT among patients. It seems the spermatozoa from semen where there were many motile spermatozoa may have higher freezing resistance. Further studies using this method in cryptozoospermic semen, testicular and epididymal spermatozoa are required.

  14. Aqueous phase catalytic conversion of agarose to 5-hydroxymethylfurfural by metal chlorides

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Lishi; Laskar, Dhrubojyoti D.; Lee, Suh-Jane; Yang, Bin

    2013-12-14

    Abstract: 5-HMF is a key intermediate for producing chemicals and fuels that can substitute for today’s petroleum-derived feedstocks. A series of metal chlorides, including NaCl, CaCl2, MgCl2, ZnCl2, CuCl2, FeCl3, and CrCl3, were comparatively investigated to catalyze agarose degradation for production of 5-HMF at temperature 180 oC, 200 oC, and 220 oC for 30 min, with catalyst concentration of 0.5% (w/w), 1% (w/w) and 5% (w/w), and substrate concentration of 2% (w/w). Our results revealed that alkali metal chlorides and alkali earth metal chlorides such as NaCl, CaCl2 and MgCl2 gave better 5-HMF yield compared with transition metal chlorides including ZnCl2, CrCl3, CuCl2 and FeCl3. 1% (w/w) MgCl2 was the more favorable catalyst for 5-HMF production from agarose, and resulted in 40.7% 5-HMF yield but no levulinic acid or lactic acid at 200 oC, 35 min. The reaction pathways of agarose degradation catalyzed by MgCl2 were also discussed.

  15. High throughput generation and trapping of individual agarose microgel using microfluidic approach

    KAUST Repository

    Shi, Yang

    2013-02-28

    Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering. © 2013 Springer-Verlag Berlin Heidelberg.

  16. A detailed study of homogeneous agarose/hydroxyapatite nanocomposites for load-bearing bone tissue.

    Science.gov (United States)

    Hu, Jingxiao; Zhu, Youjia; Tong, Hua; Shen, Xinyu; Chen, Li; Ran, Jiabing

    2016-01-01

    Agarose/hydroxyapatite (agar/HA) nanocomposites for load-bearing bone substitutes were successfully fabricated via a novel in situ precipitation method. Observation via SEM and TEM revealed that the spherical inorganic nanoparticles of approximately 50 nm were well dispersed in the organic matrix, and the crystallographic area combined closely with the amorphous area. The uniform dispersion of HA nanoparticles had prominent effect on improving the mechanical properties of the agar/HA nanocomposites (the highest elastic modulus: 1104.42 MPa; the highest compressive strength: 400.039 MPa), which proved to be potential load-bearing bone substitutes. The thermal stability of agarose and nanocomposites was also studied. The MG63 osteoblast-like cells on the composite disks displayed fusiform and polygonal morphology in the presence of HA, suggesting that the cell maturation was promoted. The results of cell proliferation and cell differentiation indicated that the cells cultured on the agar/HA composite disks significantly increased the alkaline phosphatase activity and calcium deposition. The structural role of agarose in the composite system was investigated to better understand the effect of biopolymer on structure and properties of the composites. The optimal properties were the result of a comprehensive synergy of the components. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Characterization of β-Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose

    Science.gov (United States)

    Borges, Diogo G.; Tardioli, Paulo W.; Farinas, Cristiane S.

    2014-01-01

    β-Glucosidase (BGL) is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger cultivated under solid-state fermentation (SSF) was partially purified and fully characterized in terms of optimum pH, temperature, and thermostability. The single-step purification using MANAE-agarose in a chromatographic column yielded an enzyme solution with specific activity (17.1 IU/mg protein) adequate for the characterization procedures. Electrophoresis SDS-PAGE and size-exclusion chromatography analysis resulted in an estimated molecular mass of 60 kDa. Higher enzyme activities were found in the range between 40 and 65°C and between pH 4 and 5.5, indicating an interesting characteristic for application in the hydrolysis of lignocellulosic biomass for biofuels production. Thermostability studies of purified BGL resulted in half-lives at 37°C of 56.3 h and at 50°C of 5.4 h. These results provide support for further studies of this enzyme towards revealing its potential biotechnological applications. PMID:24940510

  18. Characterization of β-Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose

    Directory of Open Access Journals (Sweden)

    Anderson Baraldo Junior

    2014-01-01

    Full Text Available β-Glucosidase (BGL is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger cultivated under solid-state fermentation (SSF was partially purified and fully characterized in terms of optimum pH, temperature, and thermostability. The single-step purification using MANAE-agarose in a chromatographic column yielded an enzyme solution with specific activity (17.1 IU/mg protein adequate for the characterization procedures. Electrophoresis SDS-PAGE and size-exclusion chromatography analysis resulted in an estimated molecular mass of 60 kDa. Higher enzyme activities were found in the range between 40 and 65°C and between pH 4 and 5.5, indicating an interesting characteristic for application in the hydrolysis of lignocellulosic biomass for biofuels production. Thermostability studies of purified BGL resulted in half-lives at 37°C of 56.3 h and at 50°C of 5.4 h. These results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

  19. Improved single-strand DNA sizing accuracy in capillary electrophoresis.

    OpenAIRE

    Rosenblum, B B; Oaks, F; Menchen, S; Johnson, B

    1997-01-01

    Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electroph...

  20. Pulsed-field gel electrophoresis (PFGE) for pathogenic Cronobacter species.

    Science.gov (United States)

    Yan, Qiongqiong; Fanning, Séamus

    2015-01-01

    Pulsed-field gel electrophoresis (PFGE) is a molecular-based subtyping strategy that uses a suitable DNA restriction endonuclease enzyme to cut genomic DNA into several large linear fragments, that can be separated based on their sizes. PFGE has been successfully applied to the subtyping of many pathogenic bacteria, including Cronobacter species, and it is commonly considered as a "gold standard" in epidemiological studies.

  1. 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.

  2. 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.

  3. 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.

  4. Self-spreading method for forming lipid bilayer on a patterned agarose gel: Toward precise lipid bilayer patterning.

    Science.gov (United States)

    Shimba, Kenta; Shoji, Kazuma; Miyamoto, Yoshitaka; Yagi, Tohru

    2017-07-01

    Forming artificial cell membranes is a suitable strategy for studying drug responses of membrane proteins. In order to form lipid bilayer with both mechanical stability and membrane protein functions, hydrogel supported bilayer has attracted attentions. Combinational use of self-extraction method for lipid bilayer formation and agarose gel patterning should realize hydrogel-supported bilayer with any shape and large area. In this study, we aimed to form a lipid bilayer on a patterned agarose gel and to characterize the membrane. First, lipid mixture was attached on an agarose gel, and lipid layers spread on the gel surface. With fluorescent observation, it is suggested that thin lipid layer was formed on the agarose gel, and their distance-dependent changes in spreading velocity was consistent with that in lipid bilayer. Next, the lipid layer was characterized with fluorescence recovery after photo breaching experiment. As a result, it is indicated that lipid molecules in the lipid layer on the agarose showed lateral diffusion, a typical characteristic of lipid bilayer. Taken together, we confirmed that lipid bilayer can be formed on the patterned agarose gel with self-spreading method. The hydrogel-supported bilayer will be a suitable tool for drug discovery.

  5. Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis.

    Science.gov (United States)

    Guihen, Elizabeth; Sisk, Garry D; Scully, Norma M; Glennon, Jeremy D

    2006-06-01

    In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.

  6. 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.

  7. Spherical agarose-coated magnetic nanoparticles functionalized with a new salen for magnetic solid-phase extraction of uranyl ion

    International Nuclear Information System (INIS)

    Serenjeh, Fariba Nazari; Hashemi, Payman; Ghiasvand, Ali Reza; Naeimi, Hossein; Zakerzadeh, Elham

    2016-01-01

    The authors describe a method for magnetic solid phase extraction of uranyl ions from water samples. It is based on the use of spherical agarose-coated magnetic nanoparticles along with magnetic field agitation. The salen type Schiff base N,N’-bis(4-hydroxysalicylidene)-1,2-phenylenediamine was synthesized from resorcinol in two steps and characterized by infrared and nucleic magnetic resonance spectroscopies. The particles were then activated by an epichlorohydrin method and functionalized with the Schiff base which acts as a selective ligand for the extraction of UO 2 (II). Following preconcentration and elution with HCl, the ions were quantified by spectrophotometry using Arsenazo III as the indicator. The effects of pH value, ionic strength and amount of the adsorbent on the extraction of UO 2 (II) were optimized by a multivariate central composite design method. Six replicate analyses under optimized conditions resulted in a recovery of 96.6 % with a relative standard deviation of 3.4 % for UO 2 (II). The detection limit of the method (at a signal-to-noise ratio of 3σ) is 10 μg L -1 . The method was successfully applied to the determination of UO 2 (II) in spiked water samples. (author)

  8. BDNF gene delivery within and beyond templated agarose multi-channel guidance scaffolds enhances peripheral nerve regeneration

    Science.gov (United States)

    Gao, Mingyong; Lu, Paul; Lynam, Dan; Bednark, Bridget; Campana, W. Marie; Sakamoto, Jeff; Tuszynski, Mark

    2016-12-01

    Objective. We combined implantation of multi-channel templated agarose scaffolds with growth factor gene delivery to examine whether this combinatorial treatment can enhance peripheral axonal regeneration through long sciatic nerve gaps. Approach. 15 mm long scaffolds were templated into highly organized, strictly linear channels, mimicking the linear organization of natural nerves into fascicles of related function. Scaffolds were filled with syngeneic bone marrow stromal cells (MSCs) secreting the growth factor brain derived neurotrophic factor (BDNF), and lentiviral vectors expressing BDNF were injected into the sciatic nerve segment distal to the scaffold implantation site. Main results. Twelve weeks after injury, scaffolds supported highly linear regeneration of host axons across the 15 mm lesion gap. The incorporation of BDNF-secreting cells into scaffolds significantly increased axonal regeneration, and additional injection of viral vectors expressing BDNF into the distal segment of the transected nerve significantly enhanced axonal regeneration beyond the lesion. Significance. Combinatorial treatment with multichannel bioengineered scaffolds and distal growth factor delivery significantly improves peripheral nerve repair, rivaling the gold standard of autografts.

  9. An optimized protocol for the production of interdelta markers in Saccharomyces cerevisiae by using capillary electrophoresis.

    Science.gov (United States)

    Tristezza, Mariana; Gerardi, Carmela; Logrieco, Antonio; Grieco, Francesco

    2009-09-01

    The amplification of genomic sequence blocks flanked by delta elements of retrotransposon origin has proved to be a very convenient method for molecular characterization of Saccharomyces cerevisiae strains. Fluorescent automated capillary electrophoresis (CE) was used to detect interdelta marker (IDM) patterns in S. cerevisiae, using the ABI Prism 3130 Genetic Analyzer. Main experimental parameters were studied and the optimal conditions for IDM amplification and samples run on the CE apparatus were determined. Fingerprints from fluorescent-labelled IDM produced using CE with the same sample analyzed by agarose electrophoresis (AE) were compared. The CE analysis was able to distinguish 43 different IDM profiles among 45 S. cerevisiae isolates with a discriminating capacity of 99.8%, whereas the AE analysis of the same samples allowed the identification of 27 different patterns (discriminatory power equal to 96%). Detection of fluorescent IDM was fast and reliable, and it facilitated data comparison. For the first time in our knowledge, the fluorescent CE proved to be well suited for IDM fingerprinting. Moreover, it could be routinely applied for the molecular differentiation of S. cerevisiae strains.

  10. DNA electrophoresis in tri-block copolymer gels--experiments and Brownian dynamics simulation

    Science.gov (United States)

    Wei, Ling; van Winkle, David H.

    2015-03-01

    The mobility of double-stranded DNA ladders in Pluronics®P105, P123 and F127, was measured by two-dimensional gel electrophoresis. Pluronics®are triblock copolymers which form gel-like phases of micelles arranged with cubic order at room temperature. A 10 base pair and a 25 base pair DNA ladder were used as samples in gel electrophoresis. The monotonically decreasing mobility with increasing length observed in the agarose separations is not observed in separations in Pluronics®. Rather, a complicated dependence of mobility on DNA length is observed, where mobility vs. length increases for short DNA molecules then decreases for longer molecules. There is also a variation of mobility with length correlated to the micelle diameter. Brownian dynamics simulations of a discrete wormlike chain model were performed to simulate short DNA molecules migrating in free solution and in a face-centered cubic matrix. By incorporating hydrodynamic interactions, the trend of simulated length-dependent mobility qualitatively agrees with experimental measurements.

  11. Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments.

    Science.gov (United States)

    Birren, B W; Lai, E; Hood, L; Simon, M I

    1989-03-01

    Conventional agarose gel electrophoresis separates DNA using a static electric field. The maximum size limit for separation of DNA by this method is about 20 kilobase pairs (kb). A number of new electrophoretic techniques which employ periodic reorientation of electric fields permit separation of DNA well beyond this size limit. We sought to determine whether the use of very fast (millisecond) field switching could improve separation of DNA in the size range of 1 to 50 kb. Additionally, we have compared the resolution obtained with each of the different field switching regimens for DNA in this size range. Switching intervals of from 0.2 to 900 ms were used with unidirectional pulsing of a single electric field, with pulsed field gels, and with field inversion gel electrophoresis. Plotting the mobility of DNA as a function of size demonstrates that under the conditions used, each of these techniques offers comparable resolution. We also have examined the separation obtained when field inversion gels are run with forward and reverse fields of equal voltage and different durations, versus using fields of equal duration and different voltages. Field inversion which uses forward and reverse fields of different voltages yields resolution which is superior to the other methods examined.

  12. A CCD-based system for the detection of DNA in electrophoresis gels by UV absorption

    International Nuclear Information System (INIS)

    Mahon, A.R.; MacDonald, J.H.; Mainwood, A.; Ott, R.J.

    1999-01-01

    A method and apparatus for the detection and quantification of large fragments of unlabelled nucleic acids in agarose gels is presented. The technique is based on ultraviolet (UV) absorption by nucleotides. A deuterium source illuminates individual sample lanes of an electrophoresis gel via an array of optical fibres. As DNA bands pass through the illuminated region of the gel the amount of UV light transmitted is reduced because of absorption by the DNA. During electrophoresis the regions of DNA are detected on-line using a UV-sensitive charge coupled device (CCD). As the absorption coefficient is proportional to the mass of DNA the technique is inherently quantitative. The mass of DNA in a region of the gel is approximately proportional to the integrated signal in the corresponding section of the CCD image. This system currently has a detection limit of less than 1.25 ng compared with 2-10 ng for the most popular conventional technique, ethidium bromide (EtBr) staining. In addition the DNA sample remains in its native state. The removal of the carcinogenic dye from the detection procedure greatly reduces associated biological hazards. (author)

  13. 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 ...

  14. 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.

  15. 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

  16. misleading presentation of haemoglobin electrophoresis data

    African Journals Online (AJOL)

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

  17. 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.

  18. Electrophoresis-mass spectrometry probe

    Science.gov (United States)

    Andresen, Brian D.; Fought, Eric R.

    1987-01-01

    The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface.

  19. Capillary electrophoresis of proteins for proteomic studies.

    Science.gov (United States)

    Manabe, T

    1999-10-01

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

  20. 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)

  1. Agarose gels

    Science.gov (United States)

    2016-11-01

    Discovered in 17th-century Japan, agar is a jelly-like substance obtained by boiling algae, and it is widely used as a gelling agent for desserts in Japanese, Indian, Philippine and Vietnamese cuisine.

  2. Hydrolysis of proteins by immobilized-stabilized alcalase-glyoxyl agarose.

    Science.gov (United States)

    Tardioli, Paulo W; Pedroche, Justo; Giordano, Raquel L C; Fernández-Lafuente, Roberto; Guisán, José M

    2003-01-01

    This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.

  3. Microneedle assisted micro-particle delivery from gene guns: experiments using skin-mimicking agarose gel.

    Science.gov (United States)

    Zhang, Dongwei; Das, Diganta B; Rielly, Chris D

    2014-02-01

    A set of laboratory experiments has been carried out to determine if micro-needles (MNs) can enhance penetration depths of high-speed micro-particles delivered by a type of gene gun. The micro-particles were fired into a model target material, agarose gel, which was prepared to mimic the viscoelastic properties of porcine skin. The agarose gel was chosen as a model target as it can be prepared as a homogeneous and transparent medium with controllable and reproducible properties allowing accurate determination of penetration depths. Insertions of various MNs into gels have been analysed to show that the length of the holes increases with an increase in the agarose concentration. The penetration depths of micro-particle were analysed in relation to a number of variables, namely the operating pressure, the particle size, the size of a mesh used for particle separation and the MN dimensions. The results suggest that the penetration depths increase with an increase of the mesh pore size, because of the passage of large agglomerates. As these particles seem to damage the target surface, then smaller mesh sizes are recommended; here, a mesh with a pore size of 178 μm was used for the majority of the experiments. The operating pressure provides a positive effect on the penetration depth, that is it increases as pressure is increased. Further, as expected, an application of MNs maximises the micro-particle penetration depth. The maximum penetration depth is found to increase as the lengths of the MNs increase, for example it is found to be 1272 ± 42, 1009 ± 49 and 656 ± 85 μm at 4.5 bar pressure for spherical micro-particles of 18 ± 7 μm diameter when we used MNs of 1500, 1200 and 750 μm length, respectively. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  4. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    Energy Technology Data Exchange (ETDEWEB)

    Cuttitta, Christina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States); Ericson, Daniel L. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260 (United States); Scalia, Alexander [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 11973-5000 (United States); Roessler, Christian G. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Teplitsky, Ella [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5215 (United States); Joshi, Karan [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); PEC University of Technology, Chandigarh (India); Campos, Olven [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414 (United States); Agarwal, Rakhi; Allaire, Marc [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Orville, Allen M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sweet, Robert M.; Soares, Alexei S., E-mail: soares@bnl.gov [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  5. Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction.

    Science.gov (United States)

    Kurien, B T; Kaufman, K M; Harley, J B; Scofield, R H

    2001-09-15

    A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70-90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit. Copyright 2001 Academic Press.

  6. Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing.

    Directory of Open Access Journals (Sweden)

    Xue Gong

    Full Text Available Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.

  7. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    Science.gov (United States)

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening. PMID:25615864

  8. Sensitivity of prestaining RNA with ethidium bromide before electrophoresis and performance of subsequent northern blots using heterologous DNA probes.

    Science.gov (United States)

    Zhao, Yun; Du, Linfang; Zhang, Nianhui

    2013-06-01

    Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde-agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.

  9. 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.

  10. 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

  11. Tailor-made cell patterning using a near-infrared-responsive composite gel composed of agarose and carbon nanotubes

    International Nuclear Information System (INIS)

    Koga, Haruka; Nakazawa, Kohji; Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi

    2013-01-01

    Micropatterning is useful for regulating culture environments. We developed a highly efficient near-infrared-(NIR)-responsive gel and established a new technique that enables cell patterning by NIR irradiation. As a new culture substratum, we designed a tissue culture plate that was coated with a composite gel composed of agarose and carbon nanotubes (CNTs). A culture plate coated with agarose only showed no response to NIR irradiation. In contrast, NIR laser irradiation induced heat generation by CNTs; this permitted local solation of the CNT/agarose gel, and consequently, selective cell-adhesive regions were exposed on the tissue culture plate. The solation area was controlled by the NIR intensity, magnification of the object lens and CNT concentration in the gel. Furthermore, we formed circular patterns of HeLa cells and linear patterns of 3T3 cells on the same culture plate through selective and stepwise NIR irradiation of the CNT/agarose gel, and we also demonstrated that individual 3T3 cells migrated along a linear path formed on the CNT/agarose gel by NIR irradiation. These results indicate that our technique is useful for tailor-made cell patterning of stepwise and/or complex cell patterns, which has various biological applications such as stepwise co-culture and the study of cell migration. (paper)

  12. 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.

  13. 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.

  14. Results concerning the analysis of the reaction products resulting from genomic dna amplification using agarose gel electrophoresis for potatoes studied old varieties

    Directory of Open Access Journals (Sweden)

    Anca BACIU

    2008-05-01

    Full Text Available The author is currently involved in collecting, making an inventory, evaluation and preservation the old varieties from the Western part of Romania. In this paper 8 potato old varieties collected during 20 years and 2 varieties from National Institute of Research and Development for potato and Sugar Beet Brasov are presented. The preservation was carried out in vivo and in vitro. Important changes were observed during this time. In our work we identified many gaps in the knowledge and understanding of the origin of transformations. We made a comparison between two big areas of potato growth: Apuseni Mountains [5] and the Maramures County [3]. In these areas the potato represents the main food in winter. This work opens opportunities for future researches in the field of political and ethical decisions for potato gene pool conservation. Soon the exchange of genetic resources will be a diplomatic issue.

  15. Pravastatin Improves Glucose Regulation and Biocompatibility of Agarose Encapsulated Porcine Islets following Transplantation into Pancreatectomized Dogs

    Directory of Open Access Journals (Sweden)

    Lawrence S. Gazda

    2014-01-01

    Full Text Available The encapsulation of porcine islets is an attractive methodology for the treatment of Type I diabetes. In the current study, the use of pravastatin as a mild anti-inflammatory agent was investigated in pancreatectomized diabetic canines transplanted with porcine islets encapsulated in agarose-agarose macrobeads and given 80 mg/day of pravastatin (n=3 while control animals did not receive pravastatin (n=3. Control animals reached preimplant insulin requirements on days 18, 19, and 32. Pravastatin-treated animals reached preimplant insulin requirements on days 22, 27, and 50. Two animals from each group received a second macrobead implant: control animals remained insulin-free for 15 and 21 days (AUC = 3003 and 5078 mg/dL/24 hr days 1 to 15 and reached preimplant insulin requirements on days 62 and 131. Pravastatin treated animals remained insulin-free for 21 and 34 days (AUC = 1559 and 1903 mg/dL/24 hr days 1 to 15 and reached preimplant insulin requirements on days 38 and 192. Total incidence (83.3% versus 64.3% and total severity (22.7 versus 18.3 of inflammation on tissue surfaces were higher in the control group at necropsy. These findings support pravastatin therapy in conjunction with the transplantation of encapsulated xenogeneic islets for the treatment of diabetes mellitus.

  16. Agarose gel-coated LPG based on two sensing mechanisms for relative humidity measurement.

    Science.gov (United States)

    Miao, Yinping; Zhang, Kaikiang; Yuam, Yujie; Liu, Bo; Zhang, Hao; Liu, Yan; Yao, Jianquan

    2013-01-01

    A relative humidity (RH) sensor based on long-period grating (LPG) with different responses is proposed by utilizing agarose gel as the sensitive cladding film. The spectral characteristic is discussed as the ambient humidity level ranges from 25% to 95% RH. Since increment of RH will result in volume expansion and refractive index increment of the agarose gel, the LPG is sensitive to applied strain and ambient refractive index; both the resonance wavelength and coupling intensity present particular responses to RH within two different RH ranges (25%-65% RH and 65%-96% RH). The coupling intensity decreases within a lower RH range while it increases throughout a higher RH range. The resonance wavelength is sensitive to the higher RH levels, and the highest sensitivity reaches 114.7 pm/% RH, and shares the same RH turning point with coupling intensity response. From a practical perspective, the proposed RH sensor would find its potential applications in high humidity level, temperature-independent RH sensing and multiparameter sensing based on wavelength/power hybrid demodulation and even static RH alarm for automatic monitoring of a particular RH value owing to the nonmonotonic RH dependence of the transmission power within the whole tested RH range.

  17. 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.

  18. Evaluation of wheat by polyacrylamide gel electrophoresis

    African Journals Online (AJOL)

    PRECIOUS

    2010-01-11

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

  19. 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

  20. 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 ...

  1. Fabrication and Optimization of a PAGATA Gel Dosimeter: Increasing the Melting Point of the PAGAT Gel Dosimeter with Agarose Additive

    Directory of Open Access Journals (Sweden)

    Bakhtiar Azadbakht

    2010-12-01

    Full Text Available Introduction: The PAGAT polymer gel dosimeter melts at 30 ˚C and even at room temperature during the summer, so it needs to be kept in a cool place such as a refrigerator. To increase the stability of the PAGAT gel, different amounts of agarose were added to the PAGAT gel composition and the PAGATA gel was manufactured. Material and Methods: The PAGATA gel vials were irradiated using a Co-60 machine. Then, the samples were evaluated using a 1.5 T Siemens MRI scanner. The ingredients of the PAGATA normoxic gel dosimeter were 4.5% N-N' methylen-bis-acrylamide, 4.5% acrylamide, 4.5% gelatine, 5 mM tetrakis (THPC, 0.01 mM hydroquinone (HQ, 0.5% agarose and 86% de-ionized water (HPLC. Results: Melting point and sensitivity of the PAGAT gel dosimeter with addition of 0.0, 0.3, 0.5, 1.0, 1.5 and 2.0% of agarose were measured, in which the melting points were increased to 30, 82, 86, 88, 89 and 90°C and their sensitivities found to be 0.113, 0.1059, 0.125, 0.122, 0.115 and 0.2  respectively. Discussion and Conclusions: Adding agarose increased the sensitivity and background R2 of the evaluated samples. The optimum amount of agarose was found to be 0.5% regarding these parameters and also the melting point of the gel dosimeter. A value of 0.5% agarose was found to be an optimum value considering the increase of sensitivity to 0.125 and melting point to 86°C but at the expense of increasing the background R2 to 4.530.

  2. 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

  3. Isoenzyme patterns of pathogenic and non-pathogenic Naegleria spp. using agarose isoelectric focusing.

    Science.gov (United States)

    De Jonckheere, J F

    1982-01-01

    Using agarose isoelectric focusing, the isoenzyme patterns of 7 different enzymes were compared in 52 Naegleria strains. The pathogenic N. fowleri was found the most homogeneous species. N. lovaniensis seems to be constituted of different types which form nevertheless a cohesive group. Within N. gruberi, large interstrain band variations were found in almost all enzyme systems. A re-examination of the taxonomic position of this species may therefore be taken into consideration. High temperature strains from Australia were confirmed to be different from N. lovaniensis. Members of a new pathogenic Naegleria sp., N. australiensis, seem to occur in Europe. Large thermophilic strains with many large pores in the cysts show identical zymograms and may constitute a new species or genus.

  4. Kinetic model for whey protein hydrolysis by alcalase multipoint-immobilized on agarose gel particles

    Directory of Open Access Journals (Sweden)

    R. Sousa Jr

    2004-06-01

    Full Text Available Partial hydrolysis of whey proteins by enzymes immobilized on an inert support can either change or evidence functional properties of the produced peptides, thereby increasing their applications. The hydrolysis of sweet cheese whey proteins by alcalase, which is multipoint-immobilized on agarose gel, is studied here. A Michaelis-Menten model that takes into account competitive inhibition by the product was fitted to experimental data. The influence of pH on the kinetic parameters in the range 6.0 to 11.0 was assessed, at 50ºC. Initial reaction-rate assays in a pHstat at different concentrations of substrate were used to estimate kinetic and Michaelis-Menten parameters, k and K M. Experimental data from long-term batch assays were used to quantify the inhibition parameter, K I. The fitting of the model to the experimental data was accurate in the entire pH range.

  5. Comparative assessment of intrinsic mechanical stimuli on knee cartilage and compressed agarose constructs.

    Science.gov (United States)

    Completo, A; Bandeiras, C; Fonseca, F

    2017-06-01

    A well-established cue for improving the properties of tissue-engineered cartilage is mechanical stimulation. However, the explicit ranges of mechanical stimuli that correspond to favorable metabolic outcomes are elusive. Usually, these outcomes have only been associated with the applied strain and frequency, an oversimplification that can hide the fundamental relationship between the intrinsic mechanical stimuli and the metabolic outcomes. This highlights two important key issues: the firstly is related to the evaluation of the intrinsic mechanical stimuli of native cartilage; the second, assuming that the intrinsic mechanical stimuli will be important, deals with the ability to replicate them on the tissue-engineered constructs. This study quantifies and compares the volume of cartilage and agarose subjected to a given magnitude range of each intrinsic mechanical stimulus, through a numerical simulation of a patient-specific knee model coupled with experimental data of contact during the stance phase of gait, and agarose constructs under direct-dynamic compression. The results suggest that direct compression loading needs to be parameterized with time-dependence during the initial culture period in order to better reproduce each one of the intrinsic mechanical stimuli developed in the patient-specific cartilage. A loading regime which combines time periods of low compressive strain (5%) and frequency (0.5Hz), in order to approach the maximal principal strain and fluid velocity stimulus of the patient-specific cartilage, with time periods of high compressive strain (20%) and frequency (3Hz), in order to approach the pore pressure values, may be advantageous relatively to a single loading regime throughout the full culture period. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

  6. Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.

    Directory of Open Access Journals (Sweden)

    Sharik R Khan

    Full Text Available The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.

  7. Photothermal Microneedle Etching: Improved Three-Dimensional Microfabrication Method for Agarose Gel for Topographical Control of Cultured Cell Communities

    Science.gov (United States)

    Moriguchi, Hiroyuki; Yasuda, Kenji

    2006-08-01

    We have developed a new three-dimensional (3D) microfabrication method for agarose gel, photothermal microneedle etching (PTMNE), by means of an improved photothermal spot heating using a focused 1064 nm laser beam for melting a portion of the agarose layer at the tip of the microneedle, where a photoabsorbent chromium layer is coated to be heated. The advantage of this method is that it allows the 3D control of the melting topography within the thick agarose layer with a 2 μm resolution, whereas conventional photothermal etching can enable only two-dimensional (2D) control on the surface of the chip. By this method, we can form the spheroid clusters of particular cells from isolated single cells without any physical contact with other cells in other chambers, which is important for measuring the community effect of the cell group from isolated single cells. When we set single cancer cells in microchambers of 100 μm in diameter, formed in a 50-μm-thick agarose layer, we observed that they grew, divided, and formed spheroid clusters of cells in each microchamber. The result indicates the potential of this method to be a fundamental technique in the research of multicellular spherical clusters of cells for checking the community effect of cells in 3D structures, such as the permeabilities of chemicals and substrates into the cluster, which is complementary to conventional 2D dish cultivation and can contribute to the cell-based screening of drugs.

  8. Specific capture, recovery and culture of cancer cells using oriented antibody-modified polystyrene chips coated with agarose film.

    Science.gov (United States)

    Jeong, Jiyun; Lee, Yeolin; Yoo, Yeongeun; Lee, Myung Kyu

    2018-02-01

    Agarose gel can be used for three dimensional (3D) cell culture because it prevents cell attachment. The dried agarose film coated on a culture plate also protected cell attachment and allowed 3D growth of cancer cells. We developed an efficient method for agarose film coating on an oxygen-plasma treated micropost polystyrene chip prepared by an injection molding process. The agarose film was modified to maleimide or Ni-NTA groups for covalent or cleavable attachment of photoactivatable Fc-specific antibody binding proteins (PFcBPs) via their N-terminal cysteine residues or 6xHis tag, respectively. The antibodies photocrosslinked onto the PFcBP-modified chips specifically captured the target cells without nonspecific binding, and the captured cells grew 3D modes on the chips. The captured cells on the cleavable antibody-modified chips were easily recovered by treatment of commercial trypsin-EDTA solution. Under fluidic conditions using an antibody-modified micropost chip, the cells were mainly captured on the micropost walls of the chip rather than on the bottom of it. The presented method will also be applicable for immobilization of oriented antibodies on various microfluidic chips with different structures. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. No Evidence of Viral Transmission following Long-Term Implantation of Agarose Encapsulated Porcine Islets in Diabetic Dogs

    Directory of Open Access Journals (Sweden)

    Lawrence S. Gazda

    2014-01-01

    Full Text Available We have previously described the use of a double coated agarose-agarose porcine islet macrobead for the treatment of type I diabetes mellitus. In the current study, the long-term viral safety of macrobead implantation into pancreatectomized diabetic dogs treated with pravastatin (n=3 was assessed while 2 dogs served as nonimplanted controls. A more gradual return to preimplant insulin requirements occurred after a 2nd implant procedure (days 148, 189, and >652 when compared to a first macrobead implantation (days 9, 21, and 21 in all macrobead implanted animals. In all three implanted dogs, porcine C-peptide was detected in the blood for at least 10 days following the first implant and for at least 26 days following the second implant. C-peptide was also present in the peritoneal fluid of all three implanted dogs at 6 months after 2nd implant and in 2 of 3 dogs at necropsy. Prescreening results of islet macrobeads and culture media prior to transplantation were negative for 13 viruses. No evidence of PERV or other viral transmission was found throughout the study. This study demonstrates that the long-term (2.4 years implantation of agarose-agarose encapsulated porcine islets is a safe procedure in a large animal model of type I diabetes mellitus.

  10. Analysis of RNA by capillary electrophoresis.

    Science.gov (United States)

    Skeidsvoll, J; Ueland, P M

    1996-09-01

    Analytical parameters known to be important for the separation of DNA by capillary electrophoresis, including gel polymer concentration, electrical field strength and temperature, were investigated and optimized for the analysis of RNA molecules from 100 to 2000 bases. Denaturation, essential to obtain uniform and identifiable peaks, was accomplished by heating the sample in 80% formamide prior to electrophoresis and the presence of 2-8 M urea in the electrophoresis buffer. Efficient separations were obtained over a wide range of electrical field strengths and temperatures using the gel polymer hydroxypropylmethylcellulose (HPMC) as separation matrix. Low HPMC concentrations (RNA (> 1000 bases) whereas higher HPMC concentrations were required for optimal separation of low molecular mass RNA. An optimized system was applicable for the separation of the predominating RNA populations (small RNA of 60-300 bases (as a group of unseparated peaks), 18S and 28S rRNA) in total RNA from a human glioma cell line. This is the first systematic investigation of electrophoresis of higher molecular mass RNA in capillaries, and motivates further studies to transfer electrophoresis of RNA to the capillary format.

  11. Design and operation of a portable scanner for high performance microchip capillary array electrophoresis.

    Science.gov (United States)

    Scherer, James R; Liu, Peng; Mathies, Richard A

    2010-11-01

    We have developed a compact, laser-induced fluorescence detection scanner, the multichannel capillary array electrophoresis portable scanner (McCAEPs) as a platform for electrophoretic detection and control of high-throughput, integrated microfluidic devices for genetic and other analyses. The instrument contains a confocal optical system with a rotary objective for detecting four different fluorescence signals, a pneumatic system consisting of two pressure/vacuum pumps and 28 individual addressable solenoid valves for control of on-chip microvalves and micropumps, four Polymerase Chain Reaction (PCR) temperature control systems, and four high voltage power supplies for electrophoresis. The detection limit of the instrument is ~20 pM for on-chip capillary electrophoresis of fluorescein dyes. To demonstrate the system performance for forensic short tandem repeat (STR) analysis, two experiments were conducted: (i) electrophoretic separation and detection of STR samples on a 96-lane microfabricated capillary array electrophoresis microchip. Fully resolved PowerPlex(®) 16 STR profiles amplified from 1 ng of 9947A female standard DNA were successfully obtained; (ii) nine-plex STR amplification, sample injection, separation, and fluorescence detection of 100-copy 9948 male standard DNA in a single integrated PCR- capillary electrophoresis microchip. These results demonstrate that the McCAEPs can be used as a versatile control and detection instrument that operates integrated microfluidic devices for high-performance forensic human identification.

  12. Tuning mechanical performance of poly(ethylene glycol) and agarose interpenetrating network hydrogels for cartilage tissue engineering.

    Science.gov (United States)

    Rennerfeldt, Deena A; Renth, Amanda N; Talata, Zsolt; Gehrke, Stevin H; Detamore, Michael S

    2013-11-01

    Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but they can be limited in cartilage tissue engineering applications due to inadequate mechanical performance. In an effort to address this limitation, our team previously reported the drastic improvement in the mechanical performance of interpenetrating networks (IPNs) of poly(ethylene glycol) diacrylate (PEG-DA) and agarose relative to pure PEG-DA and agarose networks. The goal of the current study was specifically to determine the relative importance of PEG-DA concentration, agarose concentration, and PEG-DA molecular weight in controlling mechanical performance, swelling characteristics, and network parameters. IPNs consistently had compressive and shear moduli greater than the additive sum of either single network when compared to pure PEG-DA gels with a similar PEG-DA content. IPNs withstood a maximum stress of up to 4.0 MPa in unconfined compression, with increased PEG-DA molecular weight being the greatest contributing factor to improved failure properties. However, aside from failure properties, PEG-DA concentration was the most influential factor for the large majority of properties. Increasing the agarose and PEG-DA concentrations as well as the PEG-DA molecular weight of agarose/PEG-DA IPNs and pure PEG-DA gels improved moduli and maximum stresses by as much as an order of magnitude or greater compared to pure PEG-DA gels in our previous studies. Although the viability of encapsulated chondrocytes was not significantly affected by IPN formulation, glycosaminoglycan (GAG) content was significantly influenced, with a 12-fold increase over a three-week period in gels with a lower PEG-DA concentration. These results suggest that mechanical performance of IPNs may be tuned with partial but not complete independence from biological performance of encapsulated cells. © 2013 Elsevier Ltd. All rights reserved.

  13. 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

  14. [Analysis of Cut-off Value in Screening of Thalassemia by Capillary Hemoglobin Electrophoresis for Pregnant Women from Shenzhen Region of China].

    Science.gov (United States)

    Huo, Mei; Wu, Wen-Yuan; Liu, Mei; Gan, Zhi-Biao; Mao, Wei-Yu; Lin, Rong-Yao; Liu, Ai-Qin; He, Gui-Rong

    2016-04-01

    To investigate the cut-off value in screening of thalassemia in pregnant women from Shenzhen region by capillary hemoglobin electrophoresis. The data of capillary hemoglobin electrophoresis and genetic diagnosis of thalassemia from 2122 examined prenatal women were retrospectively analyzed. Capillary hemoglobin electrophoresis and α-, β- genetic diagnosis of thalassemia were carried out for every woman. Hemoglobin electrophoresis was performed using Capillarys 2 full-automated electrophoresis instrument. Gap polymerase chain reaction and reverse dot blot were used for genetic diagnosis of thalassemia genotyping test. The cut-off value in screening of thalassemia was determined by receiver operating characteristic curve and next to analyze the value of HbA2 and HbF in screening of thalassemia using the decided cut-off value. The areas under the curve (AUC(Roc)) of HbA2 for diagnosis of α-, β- thalassemia were 0.75 and 0.981 respectively, and the AUC(Roc) of HbF for diagnosis of β-thalassemia was 0.787. When HbA2 ≤ 2.55 was taken as the cut-off value of HbA2 for diagnosis of α-thalassemia, the sensitivity, specificity, positive likelihood ratio (LR(+)) and negative likelihood ratio (LR(-)) were 89.5%, 54.8%, 1.98, 0.19 respectively. When HbA2 ≥3.9 was taken as the cut off value of HbA2 for diagnosis of β-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 96.1%, 99.8% 480.5, 0.04 respectively. When HbF ≥0.75 was taken as the cut off value of HbF for diagnosis of β-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 83.6%, 61.8% respectively. The cut-off value in screening of thalassemia by capillarys 2 full automated electrophoresis instrument is different from that of the traditional method of hemoglobin electrophoresis, such as cellulose acetate membrane electrophoresis and agarose gel electrophoresis. Each laboratory should establish their own respective cut off value.

  15. Application of Microchip Electrophoresis for Clinical Tests

    Science.gov (United States)

    Yatsushiro, Shouki; Kataoka, Masatoshi

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

  16. Contact Charge Electrophoresis: Fundamentals and Microfluidic Applications.

    Science.gov (United States)

    Bishop, Kyle J M; Drews, Aaron M; Cartier, Charles A; Pandey, Shashank; Dou, Yong

    2018-01-31

    Contact charge electrophoresis (CCEP) uses steady electric fields to drive the oscillatory motion of conductive particles and droplets between two or more electrodes. In contrast to traditional forms of electrophoresis and dielectrophoresis, CCEP allows for rapid and sustained particle motions driven by low-power dc voltages. These attributes make CCEP a promising mechanism for powering active components for mobile microfluidic technologies. This Feature Article describes our current understanding of CCEP as well as recent strategies to harness it for applications in microfluidics and beyond.

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

    NARCIS (Netherlands)

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

    2004-01-01

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

  18. Microchip capillary electrophoresis for point-of-care analysis of lithium

    NARCIS (Netherlands)

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

    Background: Microchip capillary electrophoresis (CE) is a promising method for chemical analysis of complex samples such as whole blood. We evaluated the method for point-of-care testing of lithium. Methods: Chemical separation was performed on standard glass microchip CE devices with a conductivity

  19. 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.

  20. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.

    Science.gov (United States)

    Gong, J; Traganos, F; Darzynkiewicz, Z

    1994-05-01

    In cells undergoing apoptosis (programmed cell death), a fraction of nuclear DNA is fragmented to the size equivalent of DNA in mono- or oligonucleosomes. When such DNA is analyzed by agarose gel electrophoresis it generates the characteristic "ladder" pattern of discontinuous DNA fragments. Such a pattern of DNA degradation generally serves as a marker of the apoptotic mode of cell death. We developed a simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells. The cells are prefixed in 70% ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH 7.8, and the extract is sequentially treated with RNase A and proteinase K and then subjected to electrophoresis. The ladder pattern was detected from DNA extracted from 1-2 x 10(6) HL-60 cells, of which as few as 8% were apoptotic, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy. The method is rapid and uses nontoxic reagents (no phenol, chloroform, etc.). This approach permits the analysis of DNA extracted from the very same cell population that is subjected to measurements by flow cytometry to estimate DNA ploidy, the cell cycle distribution of nonapoptotic cells, the percentage of apoptotic cells, or other parameters. Furthermore, the cells may be stored in 70% ethanol for at least several weeks before analysis without any significant DNA degradation. Treatment with ethanol also inactivates several pathogens, thereby increasing the safety of sample handling. The method is applicable to clinical samples, which can be fixed in ethanol and then stored and/or safety transported prior to analysis.

  1. Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis.

    Science.gov (United States)

    Wang, Rong; Xie, Hua; Xu, Yue-Bing; Jia, Zheng-Ping; Meng, Xian-Dong; Zhang, Juan-Hong; Ma, Jun; Wang, Juan; Wang, Xian-Hua

    2012-03-01

    The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.

  2. Comparative Studies of Two-Dimensional Electrophoresis on Galactosidase Relating to Bombyx Lectin Activity

    OpenAIRE

    加藤, 靖夫; カトウ, ヤスオ; Yasuo, Kato

    2005-01-01

    "Comparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis on the haemolymph of the domesticated silkworm, Bombyx mori and Fraction II obtained by gel filtration from the haemolymph of B. mori was performed using the 2-D mini-slab system (Atto Co.) (the first method of 2-D PAGE) and the Mini-PROTEAN mini tube gel 2-D PAGE system (Bio-Rad Laboratories, Inc.) (the second method). Moreover, two-dimensionnal electrophoresis analysis on standard β-galactosidase, grade III ...

  3. The protective effect of caffeine on DNA photosensitive damage: a gel electrophoresis

    International Nuclear Information System (INIS)

    Huang Liping; Ma Jianhua

    2009-01-01

    Agarose gel electrophoresis was performed to study interaction effect of caffeine on photosensitive injury of DNA caused by anthraquinone-2-sulphonic acid disodium (AQS), a model compound of strong photosensitizer, under 254 nm or 365nm UV irradiation Photosensitive injury of DNA induced by AQS under deoxidized condition was used as control. The results show that caffeine may resist effectively the injury effect of photosensitive damage and strong UV irradiation on DNA. The effects depend on the caffeine and AQS concentration, and irradiation time. Caffeine in concentration of 0.01-3.0 μg/μL, may prevent DNA from damage induced by UV light, but caffeine in concentration of >5.0 μg/μL accelerates the DNA damage. In particular, in the aqueous solution system of DNA, caffeine and AQS, at pH 6.25-7.35, the caffeine in concentration of 2.5-4.50 μg/μL may resist the photosensitive injury of DNA caused by AQS under the deoxidized condition and exposure by 254 nm UV for 10 min. And caffeine in concentration of 5 μg/μL would present a synergetic effect on the photosensitive injury of DNA. Possible molecular mechanism also is discussed. (authors)

  4. Detection of radiation treatment of frozen chicken using a quick microgel electrophoresis assay

    International Nuclear Information System (INIS)

    Khan, A.A.; Khan, H.M.

    2007-01-01

    DNA Comet Assay (microgel electrophoresis of single cell) is very useful and sensitive analytical technique to investigate several types of damages of DNA - especially the fragmentation. As DNA is the essential part of several foods, either animal or plant origin, therefore this method is versatile and valuable for detection of radiation treatment of several types of food. Ionizing radiation causes DNA breakage, fragmentation, base modification and single- or double-strand breaks. Therefore, the detection of fragmented DNA present in any kind or type of food can serve as the marker of detection of radiation treatment. The cells from the chicken samples (unirradiated and irradiated) were extracted in cold PBS, embedded in agarose on microscope slides, lysed for 15 minutes in 2.5 % SDS and electrophoressed at a rate of 2V /cm for 2 minutes. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA (due to radiation treatment) stretched towards the anode and cells appearing as comets. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecule of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails. Therefore, DNA comet assay provides an inexpensive, rapid and relatively simple screening method for several kinds of radiation-processed and untreated foods. (author)

  5. Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Yinfa, Ma.

    1990-12-10

    Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will be described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.

  6. Comparison of viral RNA electrophoresis and indirect ELISA methods in the diagnosis of human rotavirus infection

    International Nuclear Information System (INIS)

    Avendano, L.F.; Dubinovsky, S.

    1984-01-01

    A total of 177 stool samples from Chilean diarrhea patients under two years of age were tested for rotavirus by two methods - the indirect enzyme-linked immunosorbent assay (indirect ELISA) and viral RNA electrophoresis in agarose gels (v RNA EPH). Fifty of the specimens came from patients with acute diarrhea and 127 came from patients with protracted diarrhea. The indirect ELISA testing was performed at the National Institutes of Health in the United States: the electrophoretic testing was carried out in Santiago, Chile by the authors. The electrophoretic method detected rotavirus in 36% of the acute samples and 25% of the samples from protracted cases, while the indirect ELISA method detected rotavirus in higher percentages of samples - 46% and 38%, respectively. These results support the conclusion that v RNA EPH is a less sensitive method for detecting rotavirus than the indirect ELISA. Nevertheless, the former method's high specificity, ease of application, and low cost make it a worthwhile alternative to indirect ELISA. Thus, considering the important role played by rotavirus in infant diarrhea and the need for a diagnostic technique that can be incorporated into the routines of medical center laboratories in developing countries, there is good reason to conclude that v RNA EPH is a useful tool for studying rotavirus diarrhea. (author)

  7. 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

  8. 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 ...

  9. Preparing size markers for gel electrophoresis.

    Science.gov (United States)

    Nilsen, Timothy W

    2013-12-01

    Here we present two simple methods for preparing radiolabeled size markers for gel electrophoresis. The first procedure describes the generation of an RNA marker ladder by the alkaline hydrolysis of (32)P 5'- or 3'-end-labeled RNA. The second procedure describes the labeling of DNA fragments produced by digestion of pBR322 with the restriction enzyme MspI.

  10. 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

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

    African Journals Online (AJOL)

    Most of the appropriate SPE test requests were from clinical haematology ... 1 Division of Chemical Pathology, Department of Pathology, National Health Laboratory Service and Faculty of Medicine and Health Sciences,. Stellenbosch ... electrophoresis (IFE)) in a South African (SA) pathology laboratory setting are limited.

  12. Fractionation of liver proteins by preparative electrophoresis.

    Science.gov (United States)

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

    2004-02-01

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

  13. 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...

  14. CRUDE PROTEIN ELECTROPHORESIS OF SEEDS OF TEN

    African Journals Online (AJOL)

    A A Essiett

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

  15. 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)

  16. The Optimization of Electrophoresis on a Glass Microfluidic Chip and its Application in Forensic Science.

    Science.gov (United States)

    Han, Jun P; Sun, Jing; Wang, Le; Liu, Peng; Zhuang, Bin; Zhao, Lei; Liu, Yao; Li, Cai X

    2017-11-01

    Microfluidic chips offer significant speed, cost, and sensitivity advantages, but numerous parameters must be optimized to provide microchip electrophoresis detection. Experiments were conducted to study the factors, including sieving matrices (the concentration and type), surface modification, analysis temperature, and electric field strengths, which all impact the effectiveness of microchip electrophoresis detection of DNA samples. Our results showed that the best resolution for ssDNA was observed using 4.5% w/v (7 M urea) lab-fabricated LPA gel, dynamic wall coating of the microchannel, electrophoresis temperatures between 55 and 60°C, and electrical fields between 350 and 450 V/cm on the microchip-based capillary electrophoresis (μCE) system. One base-pair resolution could be achieved in the 19-cm-length microchannel. Furthermore, both 9947A standard genomic DNA and DNA extracted from blood spots were demonstrated to be successfully separated with well-resolved DNA peaks in 8 min. Therefore, the microchip electrophoresis system demonstrated good potential for rapid forensic DNA analysis. © 2017 American Academy of Forensic Sciences.

  17. 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.

  18. Standardization of DNA extraction from sand flies: Application to genotyping by next generation sequencing.

    Science.gov (United States)

    Casaril, Aline Etelvina; de Oliveira, Liliane Prado; Alonso, Diego Peres; de Oliveira, Everton Falcão; Gomes Barrios, Suellem Petilim; de Oliveira Moura Infran, Jucelei; Fernandes, Wagner de Souza; Oshiro, Elisa Teruya; Ferreira, Alda Maria Teixeira; Ribolla, Paulo Eduardo Martins; de Oliveira, Alessandra Gutierrez

    2017-06-01

    Standardization of the methods for extraction of DNA from sand flies is essential for obtaining high efficiency during subsequent molecular analyses, such as the new sequencing methods. Information obtained using these methods may contribute substantially to taxonomic, evolutionary, and eco-epidemiological studies. The aim of the present study was to standardize and compare two methods for the extraction of genomic DNA from sand flies for obtaining DNA in sufficient quantities for next-generation sequencing. Sand flies were collected from the municipalities of Campo Grande, Camapuã, Corumbá and Miranda, state of Mato Grosso do Sul, Brazil. Three protocols using a silica column-based commercial kit (ReliaPrep™ Blood gDNA Miniprep System kit, Promega ® ), and three protocols based on the classical phenol-chloroform extraction method (Uliana et al., 1991), were compared with respect to the yield and quality of the extracted DNA. DNA was quantified using a Qubit 2.0 fluorometer. The presence of sand fly DNA was confirmed by PCR amplification of the IVS6 region (constitutive gene), followed by electrophoresis on a 1.5% agarose gel. A total of 144 male specimens were analyzed, 72 per method. Significant differences were observed between the two methods tested. Protocols 2 and 3 of phenol-chloroform extraction presented significantly better performance than all commercial kit extraction protocols tested. For phenol-chloroform extraction, protocol 3 presented significantly better performance than protocols 1 and 2. The IVS6 region was detected in 70 of 72 (97.22%) samples extracted with phenol, including all samples for protocols 2 and 3. This is the first study on the standardization of methods for the extraction of DNA from sand flies for application to next-generation sequencing, which is a promising tool for entomological and molecular studies of sand flies. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. 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... Instruments § 862.2485 Electrophoresis apparatus for clinical use. (a) Identification. An electrophoresis apparatus for clinical use is a device intended to separate molecules or particles, including plasma...

  20. Stabilization of Candida antarctica Lipase B (CALB Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI

    Directory of Open Access Journals (Sweden)

    Sara Peirce

    2016-06-01

    Full Text Available Lipase B from Candida antarctica (CALB was immobilized on octyl agarose (OC and physically modified with polyethyleneimine (PEI in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.

  1. Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

    Directory of Open Access Journals (Sweden)

    Belinda Pingguan-Murphy

    2012-08-01

    Full Text Available OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05. The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05, indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.

  2. Mechanically tailored agarose hydrogels through molecular alloying with β-sheet polysaccharides.

    Science.gov (United States)

    Forget, Aurelien; Pique, Raphaelle-Anne; Ahmadi, Vincent; Lüdeke, Steffen; Shastri, V Prasad

    2015-01-01

    There is mounting evidence that the mechanical property of tissues provides important cues that control cell fate. However, implementation of hydrogels with tunable physicochemical properties is limited due to the challenges associated with crosslinking chemistries. It has been recently shown that mechanically well-defined injectable polysaccharide hydrogels can be engineered by switching their secondary structure from an α-helix to a β-sheet. Based on these findings, a new concept is presented to tailor the mechanical properties of agarose hydrogels via the blending with the β-sheet-rich carboxylated derivative. Using this simple strategy, gels with predictable roughness, fiber organization, and shear modulus ranging from 0.1 to 100 kPa can be formulated. Hydrogels whose mechanical properties can be precisely tailored in vivo without the recourse for chemical reactions are expected to play an important role in implementing mechanobiology paradigms in de novo tissue engineering. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Electrolysis induces pH gradients and domain orientation in agarose gels

    Science.gov (United States)

    Michelman-Ribeiro, Ariel; Nossal, Ralph; Morris, Ryan; Lange, Sarah; Kuo, Chein-Shiu; Bansil, Rama

    2006-01-01

    We have used small-angle light-scattering (SALS), microscopy, and pH measurements to study structural changes produced in unbuffered agarose gels as ions migrate under applied electric fields (3-20V/cm) . Anisotropic, bowtielike, light-scattering patterns were observed, whose development occurred more quickly at higher fields. The horizontal lobes were more pronounced at higher polymer concentration. Analysis of the SALS data with a simple model of scattering from anisotropic rods in an electric field is consistent with anisotropic rodlike domains on the order of 10-15μm in length, which align perpendicular to the electric field. The anisotropic domains in the gel reach almost the same level of orientation, regardless of the field strength. Microscope imaging revealed anisotropic domains on the same length scale, also aligned perpendicular to the field. Profiles of pH variation across the gel, measured by video photography, indicate that the anisotropic patterns appear when the H+ and OH- ions, migrating in opposite directions, meet. Calculations of pH profiles using a model based on electrodiffusion reproduce several features of measured pH profiles, including the power-law dependence on the electric field of the time at which the oppositely charged fronts meet. Ions migrating from both ends of the gel produce pH changes that are correlated with macroscopic shrinking and orientation of the gel.

  4. Mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture.

    Science.gov (United States)

    Nweke, Mauryn C; McCartney, R Graham; Bracewell, Daniel G

    2017-12-29

    Mechanical characterisation of agarose-based resins is an important factor in ensuring robust chromatographic performance in the manufacture of biopharmaceuticals. Pressure-flow profiles are most commonly used to characterise these properties. There are a number of drawbacks with this method, including the potential need for several re-packs to achieve the desired packing quality, the impact of wall effects on experimental set up and the quantities of chromatography media and buffers required. To address these issues, we have developed a dynamic mechanical analysis (DMA) technique that characterises the mechanical properties of resins based on the viscoelasticity of a 1ml sample of slurry. This technique was conducted on seven resins with varying degrees of mechanical robustness and the results were compared to pressure-flow test results on the same resins. Results show a strong correlation between the two techniques. The most mechanically robust resin (Capto Q) had a critical velocity 3.3 times higher than the weakest (Sepharose CL-4B), whilst the DMA technique showed Capto Q to have a slurry deformation rate 8.3 times lower than Sepharose CL-4B. To ascertain whether polymer structure is indicative of mechanical strength, scanning electron microscopy images were also used to study the structural properties of each resin. Results indicate that DMA can be used as a small volume, complementary technique for the mechanical characterisation of chromatography media. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  5. Preparation of colloidal gold-labeled agarose-gelatin microspherules for electron microscopic studies of phagocytosis in cultured cells.

    Science.gov (United States)

    Gao, K X; Huang, L

    1987-02-01

    Agarose-gelatin microspherules about 0.5 micron or larger are prepared with emulsification of 4% agarose-gelatin sol containing 0.2 M N-octylglucoside in an organic phase composed of cyclohexane, egg lecithin, Span 80, and ethanol, followed by extraction of lipophilic components with cyclohexane and ether. Colloidal gold particles are then introduced into microspherules using gold chloride reacting at room temperature with tannic acid in a specified concentration range. After they have been coated with bovine serum albumin or mouse IgG, colloidal gold-labeled microspherules can be readily phagocytized by mouse L-cells and P388 cells after incubation for several hours. In addition to their use as a novel marker for phagocytosis, we discuss other potential uses for these colloidal gold-labeled microspherules.

  6. Tilted hexagonal post arrays: DNA electrophoresis in anisotropic media

    Science.gov (United States)

    Chen, Zhen; Dorfman, Kevin D.

    2013-01-01

    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. PMID:23868490

  7. Hollow agarose microneedle with silver coating for intradermal surface-enhanced Raman measurements: a skin-mimicking phantom study

    Science.gov (United States)

    Yuen, Clement; Liu, Quan

    2015-06-01

    Human intradermal components contain important clinical information beneficial to the field of immunology and disease diagnosis. Although microneedles have shown great potential to act as probes to break the human skin barrier for the minimally invasive measurement of intradermal components, metal microneedles that include stainless steel could cause the following problems: (1) sharp waste production, and (2) contamination due to reuse of microneedles especially in developing regions. In this study, we fabricate agarose microneedles coated with a layer of silver (Ag) and demonstrate their use as a probe for the realization of intradermal surface-enhanced Raman scattering measurements in a set of skin-mimicking phantoms. The Ag-coated agarose microneedle quantifies a range of glucose concentrations from 5 to 150 mM inside the skin phantoms with a root-mean-square error of 5.1 mM within 10 s. The needle is found enlarged by 53.9% after another 6 min inside the phantom. The shape-changing capability of this agarose microneedle ensures that the reuse of these microneedles is impossible, thus avoiding sharp waste production and preventing needle contamination, which shows the great potential for safe and effective needle-based measurements.

  8. Combination of fibrin-agarose hydrogels and adipose-derived mesenchymal stem cells for peripheral nerve regeneration

    Science.gov (United States)

    Carriel, Víctor; Garrido-Gómez, Juan; Hernández-Cortés, Pedro; Garzón, Ingrid; García-García, Salomé; Sáez-Moreno, José Antonio; Sánchez-Quevedo, María del Carmen; Campos, Antonio; Alaminos, Miguel

    2013-04-01

    Objective. The objective was to study the effectiveness of a commercially available collagen conduit filled with fibrin-agarose hydrogels alone or with fibrin-agarose hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs) in a rat sciatic nerve injury model. Approach. A 10 mm gap was created in the sciatic nerve of 48 rats and repaired using saline-filled collagen conduits or collagen conduits filled with fibrin-agarose hydrogels alone (acellular conduits) or with hydrogels containing ADMSCs (ADMSC conduits). Nerve regeneration was assessed in clinical, electrophysiological and histological studies. Main results. Clinical and electrophysiological outcomes were more favorable with ADMSC conduits than with the acellular or saline conduits, evidencing a significant recovery of sensory and motor functions. Histological analysis showed that ADMSC conduits produce more effective nerve regeneration by Schwann cells, with higher remyelination and properly oriented axonal growth that reached the distal areas of the grafted conduits, and with intensely positive expressions of S100, neurofilament and laminin. Extracellular matrix was also more abundant and better organized around regenerated nerve tissues with ADMSC conduits than those with acellular or saline conduits. Significance. Clinical, electrophysiological and histological improvements obtained with tissue-engineered ADMSC conduits may contribute to enhancing axonal regeneration by Schwann cells.

  9. 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

  10. Capillary Electrophoresis in Food and Foodomics.

    Science.gov (United States)

    Ibáñez, Clara; Acunha, Tanize; Valdés, Alberto; García-Cañas, Virginia; Cifuentes, Alejandro; Simó, Carolina

    2016-01-01

    Quality and safety assessment as well as the evaluation of other nutritional and functional properties of foods imply the use of robust, efficient, sensitive, and cost-effective analytical methodologies. Among analytical technologies used in the fields of food analysis and foodomics, capillary electrophoresis (CE) has generated great interest for the analyses of a large number of compounds due to its high separation efficiency, extremely small sample and reagent requirements, and rapid analysis. The introductory section of this chapter provides an overview of the recent applications of capillary electrophoresis (CE) in food analysis and foodomics. Relevant reviews and research articles on these topics are tabulated including papers published in the period 2011-2014. In addition, to illustrate the great capabilities of CE in foodomics the chapter describes the main experimental points to be taken into consideration for a metabolomic study of the antiproliferative effect of carnosic acid (a natural diterpene found in rosemary) against HT-29 human colon cancer cells.

  11. 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...

  12. Metal Ions Analysis with Capillary Zone Electrophoresis.

    Science.gov (United States)

    Malik, Ashok Kumar; Aulakh, Jatinder Singh; Kaur, Varinder

    2016-01-01

    Capillary electrophoresis has recently attracted considerable attention as a promising analytical technique for metal ion separations. Significant advances that open new application areas for capillary electrophoresis in the analysis of metal species occurred based on various auxiliary separation principles. These are mainly due to complexation, ion pairing, solvation, and micellization interactions between metal analytes and electrolyte additives, which alter the separation selectivity in a broad range. Likewise, many separation studies for metal ions have been concentrated on the use of preelectrophoresis derivatization methodology. Approaches suitable for manipulation of selectivity for different metal species including metal cations, metal complexes, metal oxoanions, and organometallic compounds, are discussed, with special attention paid to the related electrophoretic system variables using illustrative examples.

  13. Contemporary sample stacking in analytical electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Malá, Zdeňka; Gebauer, Petr; Boček, Petr

    2011-01-01

    Roč. 32, č. 1 (2011), s. 116-126 ISSN 0173-0835 R&D Projects: GA ČR GA203/08/1536; GA ČR GAP206/10/1219; GA AV ČR IAA400310703 Institutional research plan: CEZ:AV0Z40310501 Keywords : biological samples * stacking * trace analysis * zone electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.303, year: 2011

  14. Capillary zone electrophoresis-mass spectrometer interface

    Science.gov (United States)

    D`Silva, A.

    1996-08-06

    A device for providing equal electrical potential between two loci unconnected by solid or liquid electrical conductors is provided. The device comprises a first electrical conducting terminal, a second electrical conducting terminal connected to the first terminal by a rigid dielectric structure, and an electrically conducting gas contacting the first and second terminals. This device is particularly suitable for application in the electrospray ionization interface between a capillary zone electrophoresis apparatus and a mass spectrometer. 1 fig.

  15. 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

  16. High temperature and low acid pretreatment and agarase treatment of agarose for the production of sugar and ethanol from red seaweed biomass.

    Science.gov (United States)

    Kim, Hee Taek; Yun, Eun Ju; Wang, Damao; Chung, Jae Hyuk; Choi, In-Geol; Kim, Kyoung Heon

    2013-05-01

    To obtain fermentable sugar from agarose, pretreatment of agarose by using acetic acid was conducted for short durations (10-30 min) at low acid concentrations (1-5% (w/v)) and high temperatures (110-130 °C). On testing the pretreated agarose by using an endo-β-agarase I (DagA), an exo-β-agarase II (Aga50D), and neoagarobiose hydrolase (NABH), we observed that the addition of the endo-type agarase did not increase the sugar yield. Use of the crude enzyme of Vibrio sp. EJY3 in combination with Aga50D and NABH including acetic acid pretreatment resulted in a 1.3-fold increase in the final reducing sugar yield (62.8% of theoretical maximum based on galactose and 3,6-anhydrogalactose in the initial agarose), compared to those obtained using Aga50D and NABH only after acetic acid pretreatment. The simultaneous saccharification and fermentation of pretreated agarose yielded ethanol of 37.1% theoretical maximum yield from galactose contained in the pretreated agarose. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. 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.

  18. IPN hydrogel nanocomposites based on agarose and ZnO with antifouling and bactericidal properties

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jingjing, E-mail: jjwang1@hotmail.com; Hu, Hongkai; Yang, Zhonglin; Wei, Jun; Li, Juan

    2016-04-01

    Nanocomposite hydrogels with interpenetrating polymer network (IPN) structure based on poly(ethylene glycol) methyl ether methacrylate modified ZnO (ZnO-PEGMA) and 4-azidobenzoic agarose (AG-N{sub 3}) were prepared by a one-pot strategy under UV irradiation. The hydrogels exhibited a highly macroporous spongelike structure, and the pore size decreased with the increase of the ZnO-PEGMA content. Due to the entanglement and favorable interactions between the two crosslinked networks, the IPN hydrogels exhibited excellent mechanical strength and light transmittance. The maximum compressive and tensile strengths of the IPN hydrogels reached 24.8 and 1.98 MPa respectively. The transparent IPN hydrogels transmitted more than 85% of visible light at all wavelengths (400–800 nm). The IPN hydrogels exhibited anti-adhesive property towards Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), and the bactericidal activity increased with the ZnO-PEGMA content. The incorporation of ZnO-PEGMA did not reduce the biocompatibility of the IPN hydrogels and all the IPN nanocomposites showed negligible cytotoxicity. The present study not only provided a facile method for preparing hydrogel nanocomposites with IPN structure but also developed a new hydrogel material which might be an excellent candidate for wound dressings. - Highlights: • IPN hydrogel nanocomposites were prepared by a one-pot strategy. • The maximum compressive and tensile strengths reached 24.8 and 1.98 MPa. • IPN hydrogels displayed excellent antibacterial activity and cytocompatibility. • This study provided a facile method for preparing IPN hydrogel nanocomposites.

  19. Electrophoresis: The Basics (by D. M. Hawcroft)

    Science.gov (United States)

    Voige, William H.

    1999-01-01

    D. M. Hawcroft. Oxford University Press: Oxford, 1997. 142 + xii pp. Index. ISBN 0-19-963563-3. $100.00. This concise monograph is one of a series on techniques in widespread use in biochemistry and cell and molecular biology. It seeks to present, in compact and readable form, the fundamentals of electrophoresis and does so very well. Both theory and practice are included, but emphasis is on the latter. Although the preface makes it clear that this book is intended for biologists, it also deserves a place in a truly complete chemistry library. The book is logically organized. Each of the nine chapters corresponds to either a step in an electrophoresis experiment (e.g., Chapter 7: Visualization of Separated Materials) or a major application (Chapter 4: The Electrophoresis of Native and Denatured Proteins). It is written as though the reader is getting ready to begin doing electrophoresis for the first time and needs a survey of the technique and its applications. A question that occurred to me repeatedly as I read through the book is: Exactly how did the author intend it to be used? One can view the book as either a text or a laboratory manual. As a resource that might be used as a supplementary text in a graduate or upper-division undergraduate course, it does an admirable job of presenting a thorough overview of modern electrophoresis. The figures and diagrams are exceptionally clear and present useful comparisons of results that can be obtained under a variety of conditions (e.g., the resolution of DNA fragments obtained with otherwise identical wedge and normal gels). Not all its explanations, however, are as cogent. It defines how the two portions of a discontinuous gel differ but fails to explain clearly how the porosity and pH differences result in the stacking effect, which is such a gel's primary advantage. Having it on hand as a laboratory manual would be much like having colleagues who are experts in all phases of electrophoresis to consult or to go to

  20. 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.

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

    Science.gov (United States)

    Erijman, Leonardo; Clegg, Robert M.

    1996-03-01

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

  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. RNA purification by preparative polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Petrov, Alexey; Wu, Tinghe; Puglisi, Elisabetta Viani; Puglisi, Joseph D

    2013-01-01

    Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length. It is commonly used to separate and purify RNA species after in vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to remove degradation products, and to purify labeled RNA species. To preserve RNA integrity following purification, RNA is usually visualized by UV shadowing or stained with ethidium bromide or SYBR green dyes. © 2013 Elsevier Inc. All rights reserved.

  5. Immobilization of the acylase from Escherichia coli on glyoxyl-agarose gives efficient catalyst for the synthesis of cephalosporins.

    Science.gov (United States)

    Estruch, Ilona; Tagliani, Auro R; Guisán, José Manuel; Fernández-Lafuente, Roberto; Alcántara, Andrés R; Toma, Lucio; Terreni, Marco

    2008-01-01

    The catalytic properties of penicillin G acylase (PGA) from Escherichia coli, when used in kinetically controlled N-acylation (kcNa) of cephalosporanic nuclei, can be strongly influenced by the moiety in 3-position of the cephem structure. In the synthesis of Cefonicid (1c), the adsorption of the cephalosporanic nucleus (7-SACA) in the PGA active site appeared sensitively increased by a positive ionic interaction between an arginine (ArgA145) in the enzyme active site and the sulphonic group of the β-lactam structure. Interestingly, when PGA was immobilized on solid supports, any effect depending on the substrate structure resulted minimized; the catalytic properties of this enzyme were affected with different outcomes depending on the type of matrix and binding chemistry. The PGA immobilized on glyoxyl-agarose (hydrophilic support activated with aldehyde groups) resulted in a good catalyst when used in kinetically controlled N-acylation of different cephalosporanic nuclei. This derivatives allow much better Vs/Vh(1) (defined as the ratio between the rate of synthesis and the rate of hydrolysis of the acylating agent) than the same enzyme immobilized on Eupergit C, an acrylic hydrophobic supports activated with epoxy groups. The synthetic performances of the Eupergit derivative versus different nuclei were always much poorer if compared with glyoxyl-agarose or the soluble protein. The use of PGA immobilized on glyoxyl-agarose allowed the development of efficient processes for the preparation of Cefazolin in high yield and purity. The results obtained in the optimization of this process are presented.

  6. Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis

    Science.gov (United States)

    Khan, Sharik R.; Kuzminov, Andrei

    2013-01-01

    Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percent of chromosomal DNA entering the gel. The degree of separation in PFG depends upon the size of DNA, as well as various conditions of electrophoresis, such as electric field strength (FS), time of electrophoresis, switch time and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that sub-chromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single strand interruptions results in artefactual decrease in molecular weight of linear DNA making accurate determination of the number of double strand breaks difficult. While breakage of nicked sub-chromosomal fragments is FS-independent, some high molecular weight sub-chromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. PMID:23770235

  7. Comparative analysis of thyroid extract gel filtration by dextran gel (Sephadex G-200) and agarose (Sepharose 6-B)

    International Nuclear Information System (INIS)

    Rosenthal, D.; Netto, M.; Silva, M.M. da; Fridman, J.

    1974-01-01

    Separation of thyroglobulin and havier proteins from crude thyroid gland extracts using molecular through agarose gel (Sepharose-6B) is done. In order to compare the separation obtained on Sephadex wiht that on Sepharose, parallel filtrations are run with extratcts from two thyroid adenomas, one 'cold' and one 'hot' nodule, and their normal contralateral tissues. On Sephadex, good separation is ibtained between the heavy proteins and thyroglobulin, separation between thyroglobulin and proteins is better ou Sephacex than on Sepharose althrough, due to the smaller diluition which the lighter fraction suffers on Sephadex, an efficient qualitative analysis is possible [pt

  8. Improving precision in gel electrophoresis by stepwisely decreasing variance components.

    Science.gov (United States)

    Schröder, Simone; Brandmüller, Asita; Deng, Xi; Ahmed, Aftab; Wätzig, Hermann

    2009-10-15

    Many methods have been developed in order to increase selectivity and sensitivity in proteome research. However, gel electrophoresis (GE) which is one of the major techniques in this area, is still known for its often unsatisfactory precision. Percental relative standard deviations (RSD%) up to 60% have been reported. In this case the improvement of precision and sensitivity is absolutely essential, particularly for the quality control of biopharmaceuticals. Our work reflects the remarkable and completely irregular changes of the background signal from gel to gel. This irregularity was identified as one of the governing error sources. These background changes can be strongly reduced by using a signal detection in the near-infrared (NIR) range. This particular detection method provides the most sensitive approach for conventional CCB (Colloidal Coomassie Blue) stained gels, which is reflected in a total error of just 5% (RSD%). In order to further investigate variance components in GE, an experimental Plackett-Burman screening design was performed. The influence of seven potential factors on the precision was investigated using 10 proteins with different properties analyzed by NIR detection. The results emphasized the individuality of the proteins. Completely different factors were identified to be significant for each protein. However, out of seven investigated parameters, just four showed a significant effect on some proteins, namely the parameters of: destaining time, staining temperature, changes of detergent additives (SDS and LDS) in the sample buffer, and the age of the gels. As a result, precision can only be improved individually for each protein or protein classes. Further understanding of the unique properties of proteins should enable us to improve the precision in gel electrophoresis.

  9. P126-T A Rapid and Sensitive Method for RNA Quality Determination on Capillary Electrophoresis Systems

    OpenAIRE

    Shah, A. B.; Karudapuram, S.; Joe, L. K.; Hauser, J.; Huynh, A. T.; Pothini, S.; Samsani, S.; Vennemeyer, E. S.; Briggs, J.; Wenz, M.; Waldron, C.; Buckelew, A.; Carver, C.; Bass, S.; Baumgartner, P.

    2007-01-01

    RNA quality is directly correlated to the success of various applications, such as microarray or real time qPCR-based gene expression analyses, cDNA library construction, Northern analyses, and RNAse protection assays, which utilize RNA samples from various organisms, tissues, cell lines, and precious biological samples. We present a novel method for examining RNA integrity and purity using capillary electrophoresis that is more cost-effective and scalable than current standard methods. This ...

  10. Investigation of the electrical properties of agarose gel: characterization of concentration using nyquist plot phase angle and the implications of a more comprehensive in vitro model of the brain

    OpenAIRE

    Pomfret, Roland; Sillay, Karl; Miranpuri, Gurwattan

    2013-01-01

    Background The electrical properties of agarose gel, namely impedance and capacitance, are relatively unexplored. Agarose gels are used as in vitro models in studies across numerous disciplines, including imaging, radiotherapy, infusion, and neurosurgery. Purpose In this study, we seek to characterize the impedance response of low concentration agarose gels by relating the gel concentrations to Nyquist Plot phase in order to establish a baseline with which to modify the response of the gel to...

  11. Gel Electrophoresis of Gold-DNA Nanoconjugates

    Directory of Open Access Journals (Sweden)

    T. Pellegrino

    2007-01-01

    Full Text Available Gold-DNA conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels. A controlled number of single-stranded DNA of different length was attached specifically via thiol-Au bonds to phosphine-stabilized colloidal gold nanoparticles. Alternatively, the surface of the gold particles was saturated with single stranded DNA of different length either specifically via thiol-Au bonds or by nonspecific adsorption. From the experimentally determined electrophoretic mobilities, estimates for the effective diameters of the gold-DNA conjugates were derived by applying two different data treatment approaches. The first method is based on making a calibration curve for the relation between effective diameters and mobilities with gold nanoparticles of known diameter. The second method is based on Ferguson analysis which uses gold nanoparticles of known diameter as reference database. Our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation, though relative changes in size upon binding of molecules can be detected with high precision. Furthermore, in this study, the specific attachment of DNA via gold-thiol bonds to Au nanoparticles is compared to nonspecific adsorption of DNA. Also, the maximum number of DNA molecules that can be bound per particle was determined.

  12. Equivalence-point electromigration acid-base titration via moving neutralization boundary electrophoresis.

    Science.gov (United States)

    Yang, Qing; Fan, Liu-Yin; Huang, Shan-Sheng; Zhang, Wei; Cao, Cheng-Xi

    2011-04-01

    In this paper, we developed a novel method of acid-base titration, viz. the electromigration acid-base titration (EABT), via a moving neutralization boundary (MNR). With HCl and NaOH as the model strong acid and base, respectively, we conducted the experiments on the EABT via the method of moving neutralization boundary for the first time. The experiments revealed that (i) the concentration of agarose gel, the voltage used and the content of background electrolyte (KCl) had evident influence on the boundary movement; (ii) the movement length was a function of the running time under the constant acid and base concentrations; and (iii) there was a good linearity between the length and natural logarithmic concentration of HCl under the optimized conditions, and the linearity could be used to detect the concentration of acid. The experiments further manifested that (i) the RSD values of intra-day and inter-day runs were less than 1.59 and 3.76%, respectively, indicating similar precision and stability in capillary electrophoresis or HPLC; (ii) the indicators with different pK(a) values had no obvious effect on EABT, distinguishing strong influence on the judgment of equivalence-point titration in the classic one; and (iii) the constant equivalence-point titration always existed in the EABT, rather than the classic volumetric analysis. Additionally, the EABT could be put to good use for the determination of actual acid concentrations. The experimental results achieved herein showed a new general guidance for the development of classic volumetric analysis and element (e.g. nitrogen) content analysis in protein chemistry. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    Science.gov (United States)

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  14. Separation of galactose, 5-hydroxymethylfurfural and levulinic acid in acid hydrolysate of agarose by nanofiltration and electrodialysis.

    Science.gov (United States)

    Kim, Jae Hyung; Na, Jeong-Geol; Yang, Ji-Won; Chang, Yong Keun

    2013-07-01

    A two-stage membrane process for the separation of galactose, 5-hydroxymethylfurfural (5-HMF) and levulinic acid (LA) has been proposed. The first step of nanofiltration (NF) is to remove 5-HMF and LA from galactose solution obtained by the hydrolysis of agarose, the main component of red algal galactan for the reduction of its microbial toxicity. 5-HMF and LA are inhibitory to fermentation but at the same time useful compounds themselves with many applications. The second step of electrodialysis (ED) is to separate 5-HMF and LA in the permeate from NF. More than 91% of 5-HMF and up to 62% of LA could be removed from agarose hydrolysate, while galactose was almost completely retained by NF. Further removal of LA was expected to be possible with no loss of galactose by operating the NF process in a diafiltration mode. 5-HMF and LA could be effectively separated from each other by ED. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. 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…

  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. 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)

  18. Electrokinetic Methods for Preparative Electrophoresis on a Chip

    NARCIS (Netherlands)

    Zalewski, D.R.

    2008-01-01

    This thesis describes research on preparative capillary electrophoresis on a chip. Capillary electrophoresis on a chip has one important drawback: the amount of an analyte obtained from a single run is very limited. Consequently, post-separation processing of the separated sample is challenging.

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

    African Journals Online (AJOL)

    Analysis of large numbers of SSR (simple sequence repeats: microsatellites) reactions can be tedious, time-consuming and expensive. The objective of this study was to report a new electrophoresis method to analyze and visualize SSR data quickly and accurately and compare it to the ability of four other electrophoresis ...

  20. 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

  1. Chiral capillary electrophoresis-mass spectrometry.

    Science.gov (United States)

    Domínguez-Vega, Elena; Crego, Antonio L; Marina, Maria Luisa

    2013-01-01

    Capillary electrophoresis-mass spectrometry (CE-MS) is a powerful analytical tool, especially in the case of chiral separations, due to the fact that it combines the high efficiency, short analysis time, and versatility of the CE with the sensitivity, selectivity, and the capacity for the identification of unknown chiral compounds offered by MS detection. This chapter describes three methodologies enabling the chiral separation of cationic and anionic compounds using different strategies, illustrating the most employed approaches used in chiral CE-MS. The first methodology uses the partial filling technique for the enantioseparation of a cationic compound using a neutral cyclodextrin. Secondly, the enantioseparation of a cationic compound using low concentrations of a neutral cyclodextrin under acidic conditions is described. Finally, a methodology for the chiral separation of an anionic compound employing low concentrations of a native cyclodextrin under basic conditions is illustrated.

  2. 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.

  3. Recent Developments in Instrumentation for Capillary Electrophoresis and Microchip-Capillary Electrophoresis

    OpenAIRE

    Felhofer, Jessica L.; Blanes, Lucas; Garcia, Carlos D.

    2010-01-01

    Over the last years there has been an explosion in the number of developments and applications of capillary electrophoresis (CE) and microchip-CE. In part, this growth has been the direct consequence of recent developments in instrumentation associated with CE. This review, which is focused on contributions published in the last five years, is intended to complement the papers presented in this special issue dedicated to Instrumentation and to provide an overview on the general trend and some...

  4. Light emitting diode, photodiode-based fluorescence detection system for DNA analysis with microchip electrophoresis.

    Science.gov (United States)

    Hall, Gordon H; Glerum, D Moira; Backhouse, Christopher J

    2016-02-01

    Electrophoretic separation of fluorescently end-labeled DNA after a PCR serves as a gold standard in genetic diagnostics. Because of their size and cost, instruments for this type of analysis have had limited market uptake, particularly for point-of-care applications. This might be changed through a higher level of system integration and lower instrument costs that can be realized through the use of LEDs for excitation and photodiodes for detection--if they provide sufficient sensitivity. Here, we demonstrate an optimized microchip electrophoresis instrument using polymeric fluidic chips with fluorescence detection of end-labeled DNA with a LOD of 0.15 nM of Alexa Fluor 532. This represents orders of magnitude improvement over previously reported instruments of this type. We demonstrate the system with an electrophoretic separation of two PCR products and their respective primers. We believe that this is the first LED-induced fluorescence microchip electrophoresis system with photodiode-based detection that could be used for standard applications of PCR and electrophoresis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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

  6. [Study on fingerprints of Citrus aurantium from different places by capillary electrophoresis].

    Science.gov (United States)

    Luo, Guang-Ming; Tan, Xiao-Hui; Xu, Li-Fang; Yang, Ya-Qin; Yang, Shi-Lin

    2008-10-01

    To develop a high performance capillary electrophoresis method for Citrus aurantium fingerprints to control its quality. The background electrolyte (BGE) was an 80 mmol x L(-1) boric acid solution containing 15 mmol x L(-1) borate. The pH of the BGE was adjusted to 9.70 with KOH solution. The detection wavelength was 201 nm and a voltage of 16 kV was applied. The sample hydrodynamic injection was 0.4 ps with a duration time of 8 sec. C. aurantium was extracted by water and a set of capillary electrophoresis fingerprints (CEFP) containing 12 co-possessing peaks was obtained. There were good similarities between the standard CEFP and each set of CEFP of C. aurantium collected from eleven different places, and their similarity coefficients were between 0.973 and 0.996. The CEFP has acceptable precision, reproducibility and stability and can be used for the quality control of C. aurantium.

  7. Determination of molecular weight of silk fibroin by non-gel sieving capillary electrophoresis.

    Science.gov (United States)

    Wei, Wei; Zhang, Yaopeng; Shao, Huili; Hu, Xuechao

    2010-01-01

    A simple non-gel sieving capillary electrophoresis (NGSCE) method was established to determine the MW of silk fibroin using CE. The background electrolyte with a pH of 8.8 was based on three components: polyethylene glycol, tris(hydroxymethyl)aminomethane, and sodium dodecyl sulfate (SDS). NGSCE showed a good linear relationship with satisfactory reproducibility between the migration time and the MW of standard proteins. It was found that the regenerated silk fibroin had an MW around 83 kDa with a wide MW distribution (MWD). This absolute value is lower than the result obtained from SDS-polyacrylamide gel electrophoresis due to the different principles of the methods, but their similar MWD shapes indicated that NGSCE could be a feasible, highly sensitive, rapid method for determination of the MW of silk fibroin.

  8. Evaluation of a microfluidics-based platform and slab electrophoresis for determination of size, integrity and quantification of in vitro transcribed RNA used as a component in therapeutic drug manufacturing.

    Science.gov (United States)

    Slagter-Jäger, Jacoba Grietje; Nicolette, Charles A; Tcherepanova, Irina Y

    2012-11-01

    Ribonucleic acid (RNA) is gaining utility as a key component of immunotherapeutics to transiently express antigens or to modulate endogenous gene expression for clinical applications. As a key ancillary component of clinical grade products, RNA requires a robust method for quality control. Here we evaluated the microfluidics based platform and slab electrophoresis for determination of integrity, concentration and size of four in vitro-transcribed RNA products with sizes of 1611, 808, 475 and 290 nucleotides (nts). Our data demonstrate that the Bioanalyzer can determine both size and integrity of the RNA, but the analysis suffers from a strong well position effect. For the RNAs tested, the integrity values obtained by the Bioanalyzer demonstrate a reverse correlation with the size of the molecule and are lower than those obtained using slab electrophoresis. Agarose gel electrophoresis produced the information on size of the RNA molecule with good precision, accuracy and reproducibility. We highlight observations which need to be taken into account when developing and qualifying a method of choice for assessment of in vitro-transcribed RNA using either approach. Copyright © 2012. Published by Elsevier B.V.

  9. Investigation of the electrical properties of agarose gel: characterization of concentration using nyquist plot phase angle and the implications of a more comprehensive in vitro model of the brain.

    Science.gov (United States)

    Pomfret, Roland; Sillay, Karl; Miranpuri, Gurwattan

    2013-07-01

    The electrical properties of agarose gel, namely impedance and capacitance, are relatively unexplored. Agarose gels are used as in vitro models in studies across numerous disciplines, including imaging, radiotherapy, infusion, and neurosurgery. In this study, we seek to characterize the impedance response of low concentration agarose gels by relating the gel concentrations to Nyquist Plot phase in order to establish a baseline with which to modify the response of the gel to simulate that of in vivo brain tissue. This information is relevant to areas such as deep brain stimulation, and could have a significant impact on in vitro model design for such studies in the future. Ten agarose gels spanning four different concentrations were subjected to impedance spectroscopy using a Model 3387 DBS electrode. Phase angles were calculated and Cartesian Nyquist plots generated from the data. Results suggest that an inverse relationship exists between agarose gel concentration and phase angle. In addition, the results indicate that agarose gel reasonably emulates a constant phase element, which portrays the electrode-electrolyte interface impedance of some equivalent circuit models of brain tissue. The data shows that agarose gel is a suitable substrate for a deep brain stimulation in vitro model, but requires modification. In the future, we plan to utilize this data to determine the modifications necessary in the current agarose gel model to make it scientifically applicable to studies of both deep brain stimulation and infusion due to their overlapping variables.

  10. Hydrolysis activities of the particle of agarose-Ce4+ complex for compounds containing phosphodiester or peptide bonds

    Science.gov (United States)

    Yu, Lina; Wang, Dongfeng; Su, Lin; Luo, Yi; Sun, Liping; Xue, Changhu

    2005-07-01

    Hydrolysis activities of PACC (particle of agarose-Ce4+ complex, newly made through double emulsification) for compounds containing phosphodiester or peptide bonds were studied. The results showed that PACC could hydrolyze organophosphorous pesticides not only in water but also in vegetable juice or tea extract. Hydrolysis rates of methamidophos, omethoate and chlorpyrifos in water are 32.39%, 27.12% and 46.62% respectively, those of chlorpyrifos and methamidophos in mung sprout juice 38.28% and 35.45% respectively, and that of chlorpyrifos in tea extract 59.76%. Hydrolysis rates of BSA (bovine serum albumin) in water and protein in tea extract by PACC increase by 54.30% and 86.46% respectively as compared with the control.

  11. Evaluation of the friction coefficient, the radial stress, and the damage work during needle insertions into agarose gels.

    Science.gov (United States)

    Urrea, Fabián A; Casanova, Fernando; Orozco, Gustavo A; García, José J

    2016-03-01

    Agarose hydrogels have been extensively used as a phantom material to mimic the mechanical behavior of soft biological tissues, e.g. in studies aimed to analyze needle insertions into the organs producing tissue damage. To better predict the radial stress and damage during needle insertions, this study was aimed to determine the friction coefficient between the material of commercial catheters and hydrogels. The friction coefficient, the tissue damage and the radial stress were evaluated at 0.2, 1.8, and 10mm/s velocities for 28, 30, and 32 gauge needles of outer diameters equal to 0.36, 0.31, and 0.23mm, respectively. Force measurements during needle insertions and retractions on agarose gel samples were used to analyze damage and radial stress. The static friction coefficient (0.295±0.056) was significantly higher than the dynamic (0.255±0.086). The static and dynamic friction coefficients were significantly smaller for the 0.2mm/s velocity compared to those for the other two velocities, and there was no significant difference between the friction coefficients for 1.8 and 10mm/s. Radial stress averages were 131.2±54.1, 248.3±64.2, and 804.9±164.3Pa for the insertion velocity of 0.2, 1.8, and 10mm/s, respectively. The radial stress presented a tendency to increase at higher insertion velocities and needle size, which is consistent with other studies. However, the damage work did not show to be a good predictor of tissue damage, which appears to be due to simplifications in the analytical model. Differently to other approaches, the method proposed here based on radial stress may be extended in future studies to quantity tissue damage in vivo along the entire needle track. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. A quality comparison of protein crystals grown under containerless conditions generated by diamagnetic levitation, silicone oil and agarose gel.

    Science.gov (United States)

    Cao, Hui-Ling; Sun, Li-Hua; Li, Jian; Tang, Lin; Lu, Hui-Meng; Guo, Yun-Zhu; He, Jin; Liu, Yong-Ming; Xie, Xu-Zhuo; Shen, He-Fang; Zhang, Chen-Yan; Guo, Wei-Hong; Huang, Lin-Jun; Shang, Peng; He, Jian-Hua; Yin, Da-Chuan

    2013-10-01

    High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

  13. Capillary electrophoresis-chemiluminescence determination of norfloxacin and prulifloxacin

    Energy Technology Data Exchange (ETDEWEB)

    Yang Zhongju; Wang Xiaoli [College of Chemistry, Beijing Normal University, Beijing 100875 (China); Qin Weidong [College of Chemistry, Beijing Normal University, Beijing 100875 (China)], E-mail: qinwd@bnu.edu.cn; Zhao Huichun [College of Chemistry, Beijing Normal University, Beijing 100875 (China)], E-mail: zhaohuichun@bnu.edu.cn

    2008-08-15

    A capillary electrophoresis (CE)-chemiluminescence (CL) method for determining norfloxacin (NFLX) and prulifloxacin (PFLX) was developed based on the enhanced CL intensity of the cerium(IV)-sulfite-fluoroquinolone (FQ) reaction sensitized by terbium(III). The separation was conducted in buffer composed of 20 mM sodium citrate, 4 mM citric acid and 10 mM sodium sulfite at pH 6.1. The CL reagent solution consisted of 2 mM cerium(IV), 4 mM terbium(III) and 1.1 mM hydrochloric acid. NFLX and PFLX were baseline separated within 11 min with detection limits (S/N = 3) of 0.057 and 0.084 {mu}g mL{sup -1}, respectively. The maximum intra- and inter-day relative standard deviations (R.S.D.s) of migration time of the analytes were less than 4.0% and 4.2%, respectively. The proposed method was applied to detect NFLX and PFLX in fortified urine sample and the results were comparable to high-performance liquid chromatography (HPLC)-UV method. Moreover, the high selectivity of the CL detection and the high-separation efficiency of CE render the method the potential of quick analyzing fluoroquinolones in real complex matrix.

  14. Detection of circular telomeric DNA without 2D gel electrophoresis.

    Science.gov (United States)

    Dlaska, Margit; Anderl, Conrad; Eisterer, Wolfgang; Bechter, Oliver E

    2008-09-01

    The end of linear chromosomes forms a lasso-like structure called the t-loop. Such t-loops resemble a DNA recombination intermediate, where the single-stranded 3' overhang is arrested in a stretch of duplex DNA. Presumably, such a t-loop can also be deleted via a recombination process. This would result in the occurrence of circular extrachromosomal telomeric DNA (t-circles), which are known to be abundantly present in immortal cells engaging the recombination-based alternative lengthening of telomeres pathway (ALT pathway). Little is known about the basic mechanism of telomeric recombination in these cells and what ultimately causes the generation of such t-circles. Current standard procedures for detecting these molecules involve 2D gel electrophoresis or electron microscopy. However, both methods are labor intense and sophisticated to perform. Here, we present a simpler, faster, and equally sensitive method for detecting t-circles. Our approach is a telomere restriction fragment assay that involves the enzymatic preservation of circular DNA with Klenow enzyme followed by Bal31 degradation of the remaining linear DNA molecules. We show that with this approach t-circles can be detected in ALT cell lines, whereas no t-circles are present in telomerase-positive cell lines. We consider our approach a valid method in which t-circle generation is the experimental readout.

  15. Capillary electrophoresis-chemiluminescence determination of norfloxacin and prulifloxacin

    International Nuclear Information System (INIS)

    Yang Zhongju; Wang Xiaoli; Qin Weidong; Zhao Huichun

    2008-01-01

    A capillary electrophoresis (CE)-chemiluminescence (CL) method for determining norfloxacin (NFLX) and prulifloxacin (PFLX) was developed based on the enhanced CL intensity of the cerium(IV)-sulfite-fluoroquinolone (FQ) reaction sensitized by terbium(III). The separation was conducted in buffer composed of 20 mM sodium citrate, 4 mM citric acid and 10 mM sodium sulfite at pH 6.1. The CL reagent solution consisted of 2 mM cerium(IV), 4 mM terbium(III) and 1.1 mM hydrochloric acid. NFLX and PFLX were baseline separated within 11 min with detection limits (S/N = 3) of 0.057 and 0.084 μg mL -1 , respectively. The maximum intra- and inter-day relative standard deviations (R.S.D.s) of migration time of the analytes were less than 4.0% and 4.2%, respectively. The proposed method was applied to detect NFLX and PFLX in fortified urine sample and the results were comparable to high-performance liquid chromatography (HPLC)-UV method. Moreover, the high selectivity of the CL detection and the high-separation efficiency of CE render the method the potential of quick analyzing fluoroquinolones in real complex matrix

  16. Sequential analysis of RNA synthesis by microchip electrophoresis.

    Science.gov (United States)

    Umemoto, Yoshihiro; Kataoka, Masatoshi; Yatsushiro, Shouki; Watanabe, Masahiro; Kido, Jun-Ichi; Kakuhata, Rei; Yamamoto, Takenori; Shinohara, Yasuo; Baba, Yoshinobu

    2009-05-01

    We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to evaluate the integrity of total RNA, for the analysis of synthesized RNA. There was little interference by DNA and/or the components of the in vitro transcription system with the microchip electrophoresis. The fluorescence intensity corresponding to the synthesized RNA increased in a time-dependent manner as to the RNA synthesis reaction on sequential analysis. A result can be obtained in 160 s and only 1/10 aliquots of samples, compared with the conventional method, are required. These results indicate the potential of microchip electrophoresis for sequential analysis of RNA synthesis.

  17. RNA conformational changes analyzed by comparative gel electrophoresis.

    Science.gov (United States)

    Eschbach, Sébastien H; Lafontaine, Daniel A

    2014-01-01

    The study of biologically relevant native RNA structures is important to understand their cellular function(s). Native gel electrophoresis provides information about such native structures in solution as a function of experimental conditions. The application of native gel electrophoresis in a comparative manner allows to obtain precise information on relative angles subtended between given pair of stems in an RNA molecule. By adapting this approach, it is possible to obtain very specific structural information such as the amplitude of dihedral angles and helical rotation. As an example, we will describe how native gel electrophoresis can be used to study the folding of the S-adenosylmethionine (SAM) sensing riboswitch.

  18. 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.

  19. An apparatus for submerged gel electrophoresis.

    Science.gov (United States)

    Kozulić, B; Heimgartner, U

    1991-11-01

    A novel apparatus for submerged gel electrophoresis is described in detail. It includes an upper buffer compartment, a lower buffer compartment, and a horizontal plate between the two compartments. The horizontal plate is a heat exchanger connected to an external heater/cooler. Buffer circulates between the two compartments through openings in the horizontal plate. In the upper compartment two separated openings are positioned on each side of the horizontal plate between the side walls and long vertical barriers. The barriers initially direct the flow of buffer and define the electric field on the sides of the upper compartment. The electric field is confined essentially into a rectangular box, defined on the ends by the end walls, on the sides by the barriers, on the bottom by the cooling plate, and on the top by the air. Since the volume of buffer is smaller in the electrode compartment than in the reservoir under the cooling plate, this design enables formation of a substantially uniform electric field without creating too high a current. To enhance uniformity of the electric field, anode and cathode consist each of two platinum wires positioned one above the other at a distance of 6 mm. The electrodes can be placed parallel to the sides and perpendicular to the buffer flow or parallel to the ends and the flow of buffer. The stream of buffer in the upper compartment is regulated by two dams, perpendicular to the long barriers, on each end of the horizontal plate.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Robotics in biomedical chromatography and electrophoresis.

    Science.gov (United States)

    Fouda, H G

    1989-08-11

    The ideal laboratory robot can be viewed as "an indefatigable assistant capable of working continuously for 24 h a day with constant efficiency". The development of a system approaching that promise requires considerable skill and time commitment, a thorough understanding of the capabilities and limitations of the robot and its specialized modules and an intimate knowledge of the functions to be automated. The robot need not emulate every manual step. Effective substitutes for difficult steps must be devised. The future of laboratory robots depends not only on technological advances in other fields, but also on the skill and creativity of chromatographers and other scientists. The robot has been applied to automate numerous biomedical chromatography and electrophoresis methods. The quality of its data can approach, and in some cases exceed, that of manual methods. Maintaining high data quality during continuous operation requires frequent maintenance and validation. Well designed robotic systems can yield substantial increase in the laboratory productivity without a corresponding increase in manpower. They can free skilled personnel from mundane tasks and can enhance the safety of the laboratory environment. The integration of robotics, chromatography systems and laboratory information management systems permits full automation and affords opportunities for unattended method development and for future incorporation of artificial intelligence techniques and the evolution of expert systems. Finally, humanoid attributes aside, robotic utilization in the laboratory should not be an end in itself. The robot is a useful tool that should be utilized only when it is prudent and cost-effective to do so.

  1. Denaturing urea polyacrylamide gel electrophoresis (Urea PAGE).

    Science.gov (United States)

    Summer, Heike; Grämer, René; Dröge, Peter

    2009-10-29

    Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method(1). The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 degrees C during the gel run allows for the separation of unstructured DNA or RNA molecules. In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions. In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol (1,2).

  2. 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).

  3. The role of electrophoresis in gene electrotransfer.

    Science.gov (United States)

    Pavlin, M; Flisar, K; Kanduser, M

    2010-07-01

    Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LV + HV and HV + LV pulses. We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced for HV + LV, while for the reversed sequence, LV + HV, there was only a minor effect of the LV pulse. For the highest plasmid concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect.

  4. 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...

  5. Determination of acid dissociation constants of warfarin and hydroxywarfarins by capillary electrophoresis.

    Science.gov (United States)

    Nowak, Paweł; Olechowska, Paulina; Mitoraj, Mariusz; Woźniakiewicz, Michał; Kościelniak, Paweł

    2015-08-10

    In this work the acid dissociation constants--pKa of warfarin and its all important oxidative metabolites have been determined by capillary electrophoresis-based methods. It has resulted in a complete description of two acid-base dissociation equilibria, yet not investigated experimentally for phase I metabolites of warfarin. The capillary electrophoresis (CE) method based on the relation between effective electrophoretic mobilities and pH has proven to be a suitable tool for pKa determination, while the spectrophotometric (CE-DAD) and the internal standard methods (IS-CE), have appeared to be promising alternative approaches. The CE-DAD approach based on the change in absorbance spectra between the acidic and basic forms is a combination between capillary electrophoresis and spectrophotometric titration, and yields very consistent values of pKa1 with CE. The IS-CE, in turn, enables an estimation of pKa1 and pKa2 from only two analytical runs, however, less accurate than CE and CE-DAD. The Debye-Hückel model has been confirmed experimentally as a good predictor of pKa values at various ionic strengths. Therefore, it has been used in determination of thermodynamic pKa1 and pKa2, referring to the zero ionic strength. The results are important from the analytical, pharmacological, and theoretical points of view. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Determination of preservatives in soft drinks by capillary electrophoresis with ionic liquids as the electrolyte additives.

    Science.gov (United States)

    Sun, Bingbing; Qi, Li; Wang, Minglin

    2014-08-01

    A capillary electrophoresis method for separating preservatives with various ionic liquids as the electrolyte additives has been developed. The performances for separation of the preservatives using five ionic liquids with different anions and different substituted group numbers on imidazole ring were studied. After investigating the influence of the key parameters on the separation (the concentration of ionic liquids, pH, and the concentration of borax), it has been found that the separation efficiency could be improved obviously using the ionic liquids as the electrolyte additives and tested preservatives were baseline separated. The proposed capillary electrophoresis method exhibited favorable quantitative analysis property of the preservatives with good linearity (r(2) = 0.998), repeatability (relative standard deviations ≤ 3.3%) and high recovery (79.4-117.5%). Furthermore, this feasible and efficient capillary electrophoresis method was applied in detecting the preservatives in soft drinks, introducing a new way for assaying the preservatives in food products. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Lifescience Database Archive (English)

    Full Text Available nheim), 20 pmol primers (5' and 3' primers), 2 units of Taq DNA polymerase (Perkin-Elmer), PCR buffer... amplified DNA products were electrophoresed on 3.0% agarose gels in 0.5 x TBE buffer at 120 V for 2 h, and ...oducts were electrophoresed on 2.0% agarose gels in 0.5 x TBE buffer at 120 V for...roducts was electrophoresed on 4.0% agarose gels in 0.5 x TBE buffer at 120 V for

  8. 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

  9. Proteome research : two-dimensional gel electrophoresis and identification methods

    National Research Council Canada - National Science Library

    Rabilloud, Thierry, 1961

    2000-01-01

    "Two-dimensional electrophoresis is the central methodology in proteome research, and the state of the art is described in detail in this text, together with extensive coverage of the detection methods available...

  10. Paper Symposium Capillary Electrophoresis and Capillary Electrochromatography of Peptides

    Czech Academy of Sciences Publication Activity Database

    Kašička, Václav (ed.)

    2003-01-01

    Roč. 24, č. 5 (2003), s. 765-913 ISSN 0173-0835 Institutional research plan: CEZ:AV0Z4055905 Keywords : capillary electrophoresis * capillary electrochromatography Subject RIV: CE - Biochemistry Impact factor: 4.040, year: 2003

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

    Science.gov (United States)

    Fountoulakis, Michael; Juranville, Jean François

    2003-02-15

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

  12. 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...

  13. 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)

  14. Purification of radiolabeled RNA products using denaturing gel electrophoresis

    OpenAIRE

    Adachi, Hironori; Yu, Yi-Tao

    2014-01-01

    This unit discusses a basic method for purification of radiolabeled RNAs using denaturing polyacrylamide gel electrophoresis. The method consists of a number of experimental procedures, including total RNA preparation from yeast cells, isolation of a specific RNA from total yeast RNA, RNA 3' terminal labeling using nucleotide (5’[32P]pCp) addition (via ligation), denaturing (8 M urea) polyacrylamide gel electrophoresis, and RNA extraction from the gel slice. Key points for achieving good elec...

  15. High performance capillary electrophoresis using Van de Graaff generator

    OpenAIRE

    Lee, Seung Jae

    2017-01-01

    Capillary electrophoresis (CE) is one of the most powerful separation technique in the field of analytical chemistry and biology. Applying high voltage to CE system is the most important factor to increase separation efficiencies and resolutions. In this thesis, I describe about the theoretical explanation of CE to understand the electrical and chemical properties for achieving high performance CE system, on-chip capillary electrophoresis with star shape geometry which can be used for precise...

  16. Determination of dioxopromethazine hydrochloride by capillary electrophoresis with electrochemiluminescence detection

    International Nuclear Information System (INIS)

    Li Yunhui; Wang Chunyan; Sun Jinying; Zhou Yongchang; You Tianyan; Wang Erkang; Fung Yingsing

    2005-01-01

    The paper presents a rapid method for the determination of dioxopromethazine hydrochloride (DPZ), an antihistamine drug, by the capillary electrophoresis with electrochemiluminescene detection (CE-ECL) using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy) 3 2+ ) reagent. This CE-ECL detection method has high sensitivity, good selectivity and reproducibility for DPZ analysis. Under the optimized conditions: separation capillary, 38 cm length (25 μm i.d.); sample injection, 10 s at 8 kV; separation voltage, 12.5 kV; running buffer, 20 mmol L -1 sodium phosphate of pH 6.0; detection potential, 1.15 V; 50 mmol L -1 of phosphate buffer (pH 7.14) containing 5 mmol L -1 of Ru(bpy) 3 2+ in ECL detection cell, the detection limit of DPZ was 0.05 μmol L -1 (S/N = 3). The linear range extended from 5 to 100 μmol L -1 . The linear curve obtained was Y = 181.62 + 9.28X with a correlation coefficient of 0.9970. The relative standard deviations of the ECL intensity and the migration time for six continuous injections of 5 μmol L -1 DPZ were 3.7% and 0.92%, respectively. The CE-ECL method was applied to analyze DPZ in real samples including tablets, rat serum and human urine, and satisfactory results were obtained without interference from samples matrix. The CE-ECL technique was proved to be a potential method for the detection of DPZ in clinic analysis

  17. Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture.

    Science.gov (United States)

    Tangtrongsup, Suwimol; Kisiday, John D

    2018-01-01

    Chondrogenesis of mesenchymal stem cells (MSCs) is induced in culture conditions that have been associated with oxidative stress, although the extent to which the oxidative environment affects differentiation and extracellular matrix (ECM) accumulation is not known. The objectives of this study were to evaluate the oxidative environment during MSCs chondrogenesis in conventional serum-free medium, and the effect of serum-supplementation on intracellular reactive oxygen species (ROS) and chondrogenesis. Young adult equine MSCs were seeded into agarose and cultured in chondrogenic medium, with or without 5% fetal bovine serum (FBS), for up to 15 days. Samples were evaluated for intracellular ROS, the antioxidant glutathione, ECM and gene expression measures of chondrogenesis, and carbonylation as an indicator of oxidative damage. Intracellular ROS increased with time in culture, and was lower in medium supplemented with FBS. Glutathione decreased ∼12-fold during early chondrogenesis (p environment during MSC chondrogenesis, and suggested that lowering ROS may be an effective approach to increase collagen accumulation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:506-514, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. First-in-Human Phase 1 Trial of Agarose Beads Containing Murine RENCA Cells in Advanced Solid Tumors

    Directory of Open Access Journals (Sweden)

    Barry H. Smith

    2016-01-01

    Full Text Available Purpose Agarose macrobeads containing mouse renal adenocarcinoma cells (RMBs release factors, suppressing the growth of cancer cells and prolonging survival in spontaneous or induced tumor animals, mediated, in part, by increased levels of myocyte-enhancing factor (MEF2D via EGFR-and AKT-signaling pathways. The primary objective of this study was to determine the safety of RMBs in advanced, treatment-resistant metastatic cancers, and then its efficacy (survival, which is the secondary objective. Methods Thirty-one patients underwent up to four intraperitoneal implantations of RMBs (8 or 16 macrobeads/kg via laparoscopy in this single-arm trial (FDA BB-IND 10091; NCT 00283075. Serial physical examinations, laboratory testing, and PET-CT imaging were performed before and three months after each implant. Results RMBs were well tolerated at both dose levels (mean 660.9 per implant. AEs were (Grade 1/2 with no treatment-related SAEs. Conclusion The data support the safety of RMB therapy in advanced-malignancy patients, and the preliminary evidence for their potential efficacy is encouraging. A Phase 2 efficacy trial is ongoing.

  19. Electrophoresis of DNA and other polyelectrolytes: Physical mechanisms

    Science.gov (United States)

    Viovy, Jean-Louis

    2000-07-01

    The dramatic recent advances in molecular biology, which have opened a new era in medicine and biotechnology, rely on improved techniques to study large molecules. Electrophoresis is one of the most important of these. Separation of DNA by size, in particular, is at the heart of genome mapping and sequencing and is likely to play an increasing role in diagnosis. This article reviews, from the point of view of a physicist, the mechanisms responsible for electrophoretic separation of polyelectrolytes. This separation is mainly performed in gels, and a wide variety of migration mechanisms can come into play, depending on the polyelectrolyte's architecture, on the electric fields applied, and on the properties of the gel. After a brief review of the thermodynamic and electrohydrodynamic principles relating to polyelectrolyte solutions, the author treats the phenomenology of electrophoresis and describes the conceptual and theoretical tools in the field. The reptation mechanisms, by which large flexible polyelectrolytes thread their way through the pores of the gel matrix, play a prominent role. Biased reptation, the extension of this model to electrophoresis, provides a very intuitive framework within which numerous physical ideas can be introduced and discussed. It has been the most popular theory in this domain, and it remains an inspiring concept for current development. There have also been important advances in experimental techniques such as single-molecule viodeomicroscopy and the development of nongel separation media and mechanisms. These, in turn, form the basis for fast-developing and innovative technologies like capillary electrophoresis, electrophoresis on microchips, and molecular ratchets.

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

    Science.gov (United States)

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

    2010-04-01

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

  1. An accessible micro-capillary electrophoresis device using surface-tension-driven flow.

    Science.gov (United States)

    Mohanty, Swomitra K; Warrick, Jay; Gorski, Jack; Beebe, David J

    2009-05-01

    We present a rapidly fabricated micro-capillary electrophoresis chip that utilizes surface-tension-driven flow for sample injection and extraction of DNA. Surface-tension-driven flow (i.e. passive pumping) [G. M. Walker et al., Lab. Chip. 2002, 2, 131-134] injects a fixed volume of sample that can be predicted mathematically. Passive pumping eliminates the need for tubing, valves, syringe pumps, and other equipment typically needed for interfacing with microelectrophoresis chips. This method requires a standard micropipette to load samples before separation, and remove the resulting bands after analysis. The device was made using liquid phase photopolymerization to rapidly fabricate the chip without the need of special equipment typically associated with the construction of microelectrophoresis chips (e.g. cleanroom) [A. K. Agarwal et al., J. Micromech. Microeng. 2006, 16, 332-340; S. K. Mohanty et al., Electrophoresis 2006, 27, 3772-3778]. Batch fabrication time for the device presented here was 1.5 h including channel coating time to suppress electroosmotic flow. Devices were constructed out of poly-isobornyl acrylate and glass. A standard microscope with a UV source was used for sample detection. Separations were demonstrated using Promega BenchTop 100 bp ladder in hydroxyl ethyl cellulose (HEC) and oligonucleotides of 91 and 118 bp were used to characterize sample injection and extraction of DNA bands. The end result was an inexpensive micro-capillary electrophoresis device that uses tools (e.g. micropipette, electrophoretic power supplies, and microscopes) already present in most labs for sample manipulation and detection, making it more accessible for potential end users.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-04-15

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

  3. Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium

    Science.gov (United States)

    Fujihara, M; Comizzoli, P; Wildt, DE; Songsasen, N

    2014-01-01

    Contents Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p cat follicle viability, whereas the latter was superior (p dog follicle survival. Likewise, dog follicle viability was enhanced (p cat, the agarose gel better (p cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange. PMID:23279476

  4. 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 and arabin......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...... and arabinose and other sugars as enzyme inducers. After electrophoresis the gels were separated into several slabs by a gel cutter. Each slab was stained for a particular enzyme. Among numerous enzyme systems tested we found useful variation in esterases (EC 3.1.1.1, EC 3.1.1.2), 3-hydroxybutyrate...

  5. Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

    Directory of Open Access Journals (Sweden)

    Satoru Kaneko

    Full Text Available Single-cell pulsed-field gel electrophoresis (SCPFGE with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.

  6. ASTRO Research Fellow Presentation - A comparison of the comet assay and pulsed-field gel electrophoresis as a predictive assay for radiosensitivity in human fibroblasts

    International Nuclear Information System (INIS)

    Sarkaria, Jann N.; Eady, John J.; Peacock, John H.; Steel, G. Gordon

    1996-01-01

    Purpose/Objective: To determine whether neutral lysis single-cell gel electrophoresis (comet assay) or pulsed-field gel electrophoresis (PFGE) can be used as a predictive assay for tissue response to radiotherapy as an alternative to clonogenic survival measurements. Materials and Methods: The comet assay has been widely used to measure DNA double strand breaks (dsb) in individual cells, and it has been suggested that it could be used as an alternative to clonogenic assays to measure radiosensitivity. Previous studies in this lab have demonstrated the ability of pulsed-field gel electrophoresis, which also measures DNA dsb, to accurately predict the radiosensitivity of a panel of fibroblasts based on determination of residual DNA dsb. As part of an ongoing study examining the relationship between fibroblast radiosensitivity and normal-tissue radiation reactions, we have compared the sensitivity and accuracy of the comet assay and PFGE on a different panel of non-transformed fibroblasts derived from breast cancer patients who developed severe radiation late effects and from case-matched controls. For the measurement of initial damage, cells were suspended in PBS and irradiated on ice for the comet assay and irradiated in agarose plugs on ice for pFGE. Residual damage was measured following irradiation of confluent cultures at 37 degree sign C and subsequent incubation for four hours prior to preparation of agarose slides and plugs. All irradiations were performed with a 59 TBq 60 Co source at a dose rate of 1.7 Gy/min. Electrophoresis was performed following neutral pH cell lysis. Comet images were captured and analyzed using Optimas software with DNA damage quantitated by the comet moment. PFGE gels were analyzed using a phosphor-image analysis system and damage was quantitated based on the percent of activity released from the well. Results: The comet assay was able to detect initial DNA damage at a threshold of 5 Gy and exhibited a linear dose

  7. Parallel analysis and orthogonal identification of N-glycans with different capillary electrophoresis mechanisms

    International Nuclear Information System (INIS)

    Feng, Hua-tao; Su, Min; Rifai, Farida Nur; Li, Pingjing; Li, Sam F.Y.

    2017-01-01

    The deep involvement of glycans or carbohydrate moieties in biological processes makes glycan patterns an important direction for the clinical and medicine researches. A multiplexing CE mapping method for glycan analysis was developed in this study. By applying different CE separation mechanisms, the potential of combined parallel applications of capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) for rapid and accurate identification of glycan was investigated. The combination of CZE and MEKC demonstrated enhancing chromatography separation capacity without the compromises of sample pre-treatment and glycan concentration. The separation mechanisms for multiplexing platform were selected based on the orthogonalities of the separation of glycan standards. MEKC method exhibited promising ability for the analysis of small GU value glycans and thus complementing the unavailability of CZE. The method established required only small amount of samples, simple instrument and single fluorescent labelling for sensitive detection. This integrated method can be used to search important glycan patterns appearing in biopharmaceutical products and other glycoproteins with clinical importance. - Highlights: • Cross-validation of analytes in complex samples was done with different CE separation mechanisms. • A simple strategy is used to confirm peak identification and extend capacity of CE separation. • The method uses small amount of sample, simple instrument and single fluorescent labeling. • Selection of mechanisms is based on orthogonalities of GU values of glycan standards. • Micellar electrokinetic chromatography was suitable for analysis of small or highly sialylated glycans.

  8. Parallel analysis and orthogonal identification of N-glycans with different capillary electrophoresis mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Hua-tao [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore); Su, Min; Rifai, Farida Nur [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); Li, Pingjing [NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore); Li, Sam F.Y., E-mail: chmlifys@nus.edu.sg [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore)

    2017-02-08

    The deep involvement of glycans or carbohydrate moieties in biological processes makes glycan patterns an important direction for the clinical and medicine researches. A multiplexing CE mapping method for glycan analysis was developed in this study. By applying different CE separation mechanisms, the potential of combined parallel applications of capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) for rapid and accurate identification of glycan was investigated. The combination of CZE and MEKC demonstrated enhancing chromatography separation capacity without the compromises of sample pre-treatment and glycan concentration. The separation mechanisms for multiplexing platform were selected based on the orthogonalities of the separation of glycan standards. MEKC method exhibited promising ability for the analysis of small GU value glycans and thus complementing the unavailability of CZE. The method established required only small amount of samples, simple instrument and single fluorescent labelling for sensitive detection. This integrated method can be used to search important glycan patterns appearing in biopharmaceutical products and other glycoproteins with clinical importance. - Highlights: • Cross-validation of analytes in complex samples was done with different CE separation mechanisms. • A simple strategy is used to confirm peak identification and extend capacity of CE separation. • The method uses small amount of sample, simple instrument and single fluorescent labeling. • Selection of mechanisms is based on orthogonalities of GU values of glycan standards. • Micellar electrokinetic chromatography was suitable for analysis of small or highly sialylated glycans.

  9. 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 and arabin......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...

  10. Dual-opposite injection capillary electrophoresis: Principles and misconceptions.

    Science.gov (United States)

    Blackney, Donna M; Foley, Joe P

    2017-03-01

    Dual-opposite injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. The electroosmotic flow (EOF) is suppressed to allow all ions to reach the detector quickly. Depending on the individual electrophoretic mobilities of the analytes of interest and the effective length that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. This review discusses the principles, applications, and limitations of dual-opposite injection capillary electrophoresis. Common misconceptions regarding DOI-CE are clarified. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Purification of radiolabeled RNA products using denaturing gel electrophoresis.

    Science.gov (United States)

    Adachi, Hironori; Yu, Yi-Tao

    2014-01-06

    This unit discusses a basic method for purification of radiolabeled RNAs using denaturing polyacrylamide gel electrophoresis. The method consists of a number of experimental procedures, including total RNA preparation from yeast cells, isolation of a specific RNA from total yeast RNA, RNA 3'-terminal labeling using nucleotide (5' [(32) P]pCp) addition (via ligation), denaturing (8 M urea) polyacrylamide gel electrophoresis, and RNA extraction from the gel slice. Key points for achieving good electrophoretic separation of RNA are also discussed. Copyright © 2014 John Wiley & Sons, Inc.

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

    Science.gov (United States)

    Duncombe, Todd A; Herr, Amy E

    2013-06-07

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

  13. Affinity Electrophoresis for Analysis of Catalytic Module-Carbohydrate Interactions

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Svensson, Birte

    2017-01-01

    Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years, carbohy......Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years...

  14. Genetic Structure Change in Harvard Vaccine Strain of Clostridium Tetani for the Period of 1990 to 2010 by Pulsed-Field Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Ahmad Sakhravi

    2013-07-01

    Full Text Available Background: PFGE facilitates the differential migration of large DNA fragments through agarose gel by constantly changing the direction of the electrical field during electrophoresis. Possibility of high difference between strains and repeatability make PFGE one of the strong molecular methods in study of bacterial strains in epidemiology. To identifying and DNA fingerprinting of vaccine strain of Clostridium tetani by PFGE technique. Also, possibility of genotyping profile changes in frequency of vaccine strain of C. tetani during the period of 1990 to 2011.Materials and Methods: The vaccine strain of C. tetani was provided by Razi Vaccine and Serum Research Institute in Karaj. The seeds were inoculated into Columbia blood agar and grown for 72 h. The cultures were incubated at 35°C in anaerobic conditions. The PFGE analyses were performed using genomic DNA digested with the restriction enzyme SmaI. The electrophoresis analyses were carried out on a CHEF DR III apparatus (Bio-Rad and band patterns obtained were then analyzed.Results: The PFGE profile obtained from vaccine strain during a period of more than two decades revealed no remarkable genetic changes and mutations. This type of analysis provides detailed data useful for surveillance of vaccine strains and isolates as well as for the selection of certain predominant profiles for further investigation.Conclusion: This study showed no considerable change in chromosomal genome of Harvard, the vaccine strain. It is therefore concluded that the vaccine produced by Razi Institute had evidently no alteration or modification in accordance to PFGE profile analysis during a period of more than two decades.

  15. Paraoxonase activity against nerve gases measured by capillary electrophoresis and characterization of human serum paraoxonase (PON1) polymorphism in the coding region (Q192R).

    Science.gov (United States)

    Kanamori-Kataoka, Mieko; Seto, Yasuo

    2009-02-01

    An analytical method for determining paraoxonase activity against sarin, soman and VX was established. We used capillary electrophoresis to measure directly the hydrolysis products: alkyl methylphosphonates. After enzymatic reaction of human serum paraoxonase (PON1) with nerve gas, substrate was removed with dichloromethane, and alkyl methylphoshphonates were quantified by capillary electrophoresis of reversed osmotic flow using cationic detergent and sorbic acid. This method was applied to the characterization of human serum PON1 polymorphism for nerve gas hydrolytic activity in the coding region (Q192R). PON1-192 and PON1-55 genotypes were determined by their gel electrophoretic fragmentation pattern with restriction enzymes after polymerase chain reaction (PCR) of blood leukocyte genomic DNA. Frequencies of genotypes among 63 members of our institutes with PON1-192 and PON1-55 were 9.5% ((192)QQ), 30.1% ((192)QR) and 44.4% ((192)RR), and 82.5% ((55)LL), 17.5% ((55)LM) and 0% ((55)MM), respectively. (192)Q and (192)R enzymes were purified from the respective genotype human plasma, using blue agarose affinity chromatography and diethyl amino ethane (DEAE) anion exchange chromatography. V(max) and K(m) were measured using Lineweaver-Burk plots for hydrolytic activities against sarin, soman and VX at pH 7.4 and 25 degrees C. For sarin and soman, the V(max) for (192)Q PON1 were 3.5- and 1.5-fold higher than those for (192)R PON1; and k(cat)/K(m) for (192)Q PON1 were 1.3- and 2.8-fold higher than those for (192)R PON1. For VX, there was little difference in V(max) and k(cat)/K(m) between (192)Q and (192)R PON1, and VX hydrolyzing activity was significantly lower than those for sarin and soman. PON1 hydrolyzed sarin and soman more effectively than paraoxon.

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

    Science.gov (United States)

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

    2007-11-01

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

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

    African Journals Online (AJOL)

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

  18. Separation and quantification of cellulases and hemicellulases by capillary electrophoresis

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Kutter, Jörg Peter; Olsson, Lisbeth

    2003-01-01

    . 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...

  19. Gel Electrophoresis and Fluorescamine Methods for the Detection of ...

    African Journals Online (AJOL)

    were the most proteolysed, indicating that the high temperatures (110, 120, 130 and 142 ˚C for 2s) lowered proteolysis through inactivation of heat resistant native enzymes possibly plasmin and hence decreased milk's susceptibility to spoilage. Key words: proteolysis, fluorescamine, gel electrophoresis, plasmin, isoelectric ...

  20. Explorative data analysis of two-dimensional electrophoresis gels

    DEFF Research Database (Denmark)

    Schultz, J.; Gottlieb, D.M.; Petersen, Marianne Kjerstine

    2004-01-01

    Methods for classification of two-dimensional (2-DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two-dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening...

  1. 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, ...

  2. Electrophoresis in charge-stabilized colloidal cluster phases

    NARCIS (Netherlands)

    Groenewold, J.; Zhang, T.; Kegel, W.K.

    2011-01-01

    The reversible properties of cluster phases have been described by theories that invoke Coulomb interactions as a stabilizing mechanism.What is lacking so far is direct measurement of these charges. This contribution aims at predicting what to expect if electrophoresis measurements were to be

  3. A new electrophoresis technique to separate microsatellite alleles*

    African Journals Online (AJOL)

    GREGORY

    2009-06-03

    Jun 3, 2009 ... Analysis of large numbers of SSR (simple sequence repeats: microsatellites) reactions can be tedious, time-consuming and expensive. The objective of this study was to report a new electrophoresis method to analyze and visualize SSR data quickly and accurately and compare it to the ability of four other.

  4. 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

  5. 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

  6. Capillary electrophoresis in the analysis of biologically important thiols

    Czech Academy of Sciences Publication Activity Database

    Lačná, J.; Kubáň, Petr; Foret, František

    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

  7. Acetic acid denaturing for RNA capillary polymer electrophoresis.

    Science.gov (United States)

    Sumitomo, Keiko; Sasaki, Motoyasu; Yamaguchi, Yoshinori

    2009-05-01

    A strong denaturant to cleave intramolecular hydrogen bonds in RNA is required for RNA size separation in a small sample volume (RNA and the RNA separation performance was dramatically improved by capillary electrophoresis with a sieving matrix containing acetic acid. We revealed that the denaturing ability of 2.0 M acetic acid was stronger than that of either 2.5 M formaldehyde or 7.0 M urea by estimating DNA melting temperature. Consequently, we suggested "in-capillary denaturing polymer electrophoresis" as the RNA size separation methodology to simultaneously denature and separate RNA in a small sample volume without conventional in vitro sample preparation before electrophoresis. The baseline separation of RNA with a size of 100-10,000 nt was achieved in 25 min by "in-capillary denaturing polymer electrophoresis" with the running buffer containing 2.0 M acetic acid. The resolution and the theoretical plates of RNA separation peaks were larger than those of the RNA separation in a conventional CGE with in vitro sample preparation by 7.0 M urea. In addition, we detected RNA peaks from the nucleic acids extracted from NIH 3T3 cells without DNase enzyme treatment.

  8. Starch gel electrophoresis of conifer seeds: a laboratory manual

    Science.gov (United States)

    M. Thompson Conkle; Paul D. Hodgskiss; Lucy B. Nunnally; Serena C. Hunter

    1982-01-01

    This manual describes fast, low-cost biochemical procedures for separating enzymes representing numerous genes of forest trees. During electrophoresis the mixture of enzymes from a megagametophyte or embryo of a germinated seed separates in a gel. Specific stains applied to gel slices locate each enzyme. These procedures expand on those developed for crops research....

  9. 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

  10. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to determine apoptosis induced by siRNA in Colo 320 cells. When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a ...

  11. 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

  12. hiv and serum protein electrophoresis patterns in kwazulu

    African Journals Online (AJOL)

    2011-04-01

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

  13. 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

  14. 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 ...

  15. 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 ...

  16. 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 ...

  17. 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 ...

  18. 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

  19. Analysis of branched nucleic acid structure using comparative gel electrophoresis.

    Science.gov (United States)

    Lilley, David M J

    2008-02-01

    Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.

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

    Science.gov (United States)

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

    1982-02-01

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

  2. RiboCAT: a new capillary electrophoresis data analysis tool for nucleic acid probing.

    Science.gov (United States)

    Cantara, William A; Hatterschide, Joshua; Wu, Weixin; Musier-Forsyth, Karin

    2017-02-01

    Chemical and enzymatic probing of RNA secondary structure and RNA/protein interactions provides the basis for understanding the functions of structured RNAs. However, the ability to rapidly perform such experiments using capillary electrophoresis has been hampered by relatively labor-intensive data analysis software. While these computationally robust programs have been shown to calculate residue-specific reactivities to a high degree of accuracy, they often require time-consuming manual intervention and lack the ability to be easily modified by users. To alleviate these issues, RiboCAT (Ribonucleic acid capillary-electrophoresis analysis tool) was developed as a user-friendly, Microsoft Excel-based tool that reduces the need for manual intervention, thereby significantly shortening the time required for data analysis. Features of this tool include (i) the use of an Excel platform, (ii) a method of intercapillary signal alignment using internal size standards, (iii) a peak-sharpening algorithm to more accurately identify peaks, and (iv) an open architecture allowing for simple user intervention. Furthermore, a complementary tool, RiboDOG (RiboCAT data output generator) was designed to facilitate the comparison of multiple data sets, highlighting potential inconsistencies and inaccuracies that may have occurred during analysis. Using these new tools, the secondary structure of the HIV-1 5' untranslated region (5'UTR) was determined using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), matching the results of previous work. © 2017 Cantara et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  3. Radioautography and fluorography applied to the detection of radioactive compounds separated by electrophoresis and chromatography

    International Nuclear Information System (INIS)

    Simonnet, Gerard; Combe, Jose

    1976-01-01

    Radioautography permits the location of radioactive compounds on a wide variety of supporting media after electrophoresis or chromatography: paper, a thin layer of silica gel or polyacrylamide gel. Latent images are obtained by applying an appropriate photographic film against the gel or paper or plaque in question and leaving them for a sufficient time. The latent image is then rendered visible by standard photographic development, which results in black spots on the film corresponding to radioactive regions on the support. The use of a particular radioactive tracer implies the use of electrophoresis and chromatography in order to control the radiochemical purity of the product, and thus the validity of the results obtained. Radiolysis products, arising from chemical degradation of the product provoked by the radiation emitted, are impurities which assume a greater importance with increasing specific radioactivities. In the case of 3 H-thymidine of specific activity greater than 5 or 10 mCi/mmole, for example, the incidence of radiolysis is such that after two months of storage the product is totally inutilisable

  4. Electro-driven extraction of polar compounds using agarose gel as a new membrane: Determination of amino acids in fruit juice and human plasma samples.

    Science.gov (United States)

    Sedehi, Samira; Tabani, Hadi; Nojavan, Saeed

    2018-03-01

    In this work, polypropylene hollow fiber was replaced by agarose gel in conventional electro membrane extraction (EME) to develop a novel approach. The proposed EME method was then employed to extract two amino acids (tyrosine and phenylalanine) as model polar analytes, followed by HPLC-UV. The method showed acceptable results under optimized conditions. This green methodology outperformed conventional EME, and required neither organic solvents nor carriers. The effective parameters such as the pH values of the acceptor and the donor solutions, the thickness and pH of the gel, the extraction voltage, the stirring rate, and the extraction time were optimized. Under the optimized conditions (acceptor solution pH: 1.5; donor solution pH: 2.5; agarose gel thickness: 7mm; agarose gel pH: 1.5; stirring rate of the sample solution: 1000rpm; extraction potential: 40V; and extraction time: 15min), the limits of detection and quantification were 7.5ngmL -1 and 25ngmL -1 , respectively. The extraction recoveries were between 56.6% and 85.0%, and the calibration curves were linear with correlation coefficients above 0.996 over a concentration range of 25.0-1000.0ngmL -1 for both amino acids. The intra- and inter-day precisions were in the range of 5.5-12.5%, and relative errors were smaller than 12.0%. Finally, the optimized method was successfully applied to preconcentrate, clean up, and quantify amino acids in watermelon and grapefruit juices as well as a plasma sample, and acceptable relative recoveries in the range of 53.9-84.0% were obtained. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Simple agarose micro-confinement array and machine-learning-based classification for analyzing the patterned differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Nobuyuki Tanaka

    Full Text Available The geometrical confinement of small cell colonies gives differential cues to cells sitting at the periphery versus the core. To utilize this effect, for example to create spatially graded differentiation patterns of human mesenchymal stem cells (hMSCs in vitro or to investigate underpinning mechanisms, the confinement needs to be robust for extended time periods. To create highly repeatable micro-fabricated structures for cellular patterning and high-throughput data mining, we employed here a simple casting method to fabricate more than 800 adhesive patches confined by agarose micro-walls. In addition, a machine learning based image processing software was developed (open code to detect the differentiation patterns of the population of hMSCs automatically. Utilizing the agarose walls, the circular patterns of hMSCs were successfully maintained throughout 15 days of cell culture. After staining lipid droplets and alkaline phosphatase as the markers of adipogenic and osteogenic differentiation, respectively, the mega-pixels of RGB color images of hMSCs were processed by the software on a laptop PC within several minutes. The image analysis successfully showed that hMSCs sitting on the more central versus peripheral sections of the adhesive circles showed adipogenic versus osteogenic differentiation as reported previously, indicating the compatibility of patterned agarose walls to conventional microcontact printing. In addition, we found a considerable fraction of undifferentiated cells which are preferentially located at the peripheral part of the adhesive circles, even in differentiation-inducing culture media. In this study, we thus successfully demonstrated a simple framework for analyzing the patterned differentiation of hMSCs in confined microenvironments, which has a range of applications in biology, including stem cell biology.

  6. 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

  7. 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

  8. Advancements to the theory of free solution electrophoresis of polyelectrolytes

    Science.gov (United States)

    McCormick, Laurette

    Capillary electrophoresis (CE) is the workhorse of countless analytical laboratories and is used routinely in various industries including pharmaceutical, forensic and clinical applications. Basically, CE is a method for separating charged molecular species in a buffer-filled capillary by the application of an electric field; the analytes move from one end of the capillary to the detector at the other end at speeds determined by their charge, size and shape. Generally, in free solution CE uniformly charged polyelectrolytes (such as DNA) are free-draining, meaning that their speed is independent of their size. Hence, until recently, a gel or other sieving medium has been necessary for the separation of polyelectrolytes; however, modifying uniformly charged polymers on the molecular level, via conjugation to uncharged polymers, allows for separation in free solution CE. In this thesis, advancements to the theory of free solution electrophoresis of polyelectrolytes, in particular, to the theories for two new free solution electrophoresis methods relying on conjugation, are presented. The first method, called End Labelled Free Solution Electrophoresis (ELFSE), can be used to sequence DNA, a negatively charged polymer in solution. Two different means of improving the resolution of ELFSE are predicted, one based on the molecular end effect, the other based on using a controlled electro-osmotic flow. In addition, a theory for the segregation of the DNA and label coils in ELFSE is presented. The second method is called Free Solution Conjugate Electrophoresis (FSCE); it allows for characterization of a sample of neutral polymers differing in length. The relevant theory, developed herein, elucidates how to accurately determine the molar mass distribution of the sample through FSCE measurements. In addition, supporting theories are developed that clarify the correct equation for the diffusion coefficient of molecules undergoing free solution electrophoresis, as well as

  9. RNA quality in frozen breast cancer samples and the influence on gene expression analysis--a comparison of three evaluation methods using microcapillary electrophoresis traces.

    Science.gov (United States)

    Strand, Carina; Enell, Johan; Hedenfalk, Ingrid; Fernö, Mårten

    2007-05-22

    Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping. Comparing the methods pair-wise, Manual vs. Ratio showed concordance (good vs. degraded RNA) in 20/24, Manual vs. RIN in 23/24, and Ratio vs. RIN in 21/24 samples. All three methods were concordant in 20/24 samples. The comparison between RNA quality and gene expression analysis showed that pieces from the same tumor and with good RNA quality clustered together in most cases, whereas those with poor quality often clustered apart. The number of samples clustering in an unexpected manner was lower for the Manual (n = 1) and RIN methods (n = 2) as compared to the Ratio method (n = 5). Assigning the data into two groups, RIN > or = 6 or RIN RNA became degraded. Ontological mapping using GoMiner (p or = 3 genes changed) revealed deoxyribonuclease activity, collagen, regulation of cell adhesion, cytosolic ribosome, and NADH dehydrogenase activity, to be the five categories most affected by RNA quality

  10. RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

    Directory of Open Access Journals (Sweden)

    Hedenfalk Ingrid

    2007-05-01

    Full Text Available Abstract Background Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1 Manual method = subjective evaluation of the electropherogram, 2 Ratio Method = the ratio between the 28S and 18S peaks, and 3 RNA integrity number (RIN method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping. Results Comparing the methods pair-wise, Manual vs. Ratio showed concordance (good vs. degraded RNA in 20/24, Manual vs. RIN in 23/24, and Ratio vs. RIN in 21/24 samples. All three methods were concordant in 20/24 samples. The comparison between RNA quality and gene expression analysis showed that pieces from the same tumor and with good RNA quality clustered together in most cases, whereas those with poor quality often clustered apart. The number of samples clustering in an unexpected manner was lower for the Manual (n = 1 and RIN methods (n = 2 as compared to the Ratio method (n = 5. Assigning the data into two groups, RIN ≥ 6 or RIN i.e. when the RNA became degraded. Ontological mapping using GoMiner (p ≤ 0.05; ≥ 3 genes changed revealed deoxyribonuclease activity, collagen, regulation of cell adhesion, cytosolic ribosome, and NADH dehydrogenase

  11. Imaging Catalytic Surfaces by Multiplexed Capillary Electrophoresis With Absorption Detection

    Energy Technology Data Exchange (ETDEWEB)

    Christodoulou, Michael [Iowa State Univ., Ames, IA (United States)

    2002-01-01

    A new technique for in situ imaging and screening heterogeneous catalysts by using multiplexed capillary electrophoresis with absorption detection was developed. By bundling the inlets of a large number of capillaries, an imaging probe can be created that can be used to sample products formed directly from a catalytic surface with high spatial resolution. In this work, they used surfaces made of platinum, iron or gold wires as model catalytic surfaces for imaging. Various shapes were recorded including squares and triangles. Model catalytic surfaces consisting of both iron and platinum wires in the shape of a cross were also imaged successfully. Each of the two wires produced a different electrochemical product that was separated by capillary electrophoresis. Based on the collected data they were able to distinguish the products from each wire in the reconstructed image.

  12. Analytical separations of lanthanides and actinides by capillary electrophoresis.

    Science.gov (United States)

    Janos, Pavel

    2003-06-01

    The separation of lanthanide and actinide elements belongs to one of the most challenging tasks of the separation science, due to a great similarity in their physical and chemical properties. The electrophoretic separation can be accomplished in the presence of suitable complex-forming agents, from which alpha-hydroxyisobutyric acid (HIBA) has been used most often. In the most effective capillary electrophoretic mode--capillary zone electrophoresis (CZE)--a complete separation of lanthanide ions can be accomplished within a few minutes. Various electrophoretic methods can be relatively easily adopted for the determinations of individual lanthanide elements in certain kinds of technical materials, concentrates, precursors, etc., where the high speed and low costs of analysis characteristics of capillary electrophoresis (CE) may be advantageously exploited. Electrophoretic techniques may also be employed for speciation studies, especially for examinations of the behavior of actinides in the environment.

  13. 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.

  14. Serum Protein Electrophoresis in Dogs With Intestinal Parasites

    OpenAIRE

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

    1999-01-01

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

  15. 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

  16. 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.

  17. 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...

  18. 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...

  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. The coupling of capillary electrophoresis-inductively coupled plasma mass spectrometer as a speciation instrument for actinides at trace level

    International Nuclear Information System (INIS)

    Delorme, A.

    2004-01-01

    An interface between the separation technique (capillary electrophoresis) and the analytical technique (Inductively Coupled Plasma - Mass Spectrometer) was developed. In that sense, bibliographic and parametric studies allowed to define necessary conditions for the good working of both techniques. The results obtained led to the realisation of an interface capillary electrophoresis / ICP-MS (CE / ICP-MS). This one was experimentally validated on classical separations (alkalis / earth-alkalis and lanthanides) and the detection limit of the analytical system was determined equal to 4 x 10 -11 mol.L -1 for plutonium. This result exhibits a gain in detection limit of a factor higher than 10 4 compared to the capillary electrophoresis in standard detection (UV). The studies were made in order to check the capacity of the CE / ICP-MS coupling as a speciation instrument for actinides at trace level and to define the associated analytical procedures. The coupling turned out to be a suited instrument for the determination of absolute electrophoretic mobilities at infinite dilution (physico-chemical property which allows to predict the migration time of an ion under an electrical field in a given electrolyte), for the determination of thermodynamic constants and for the separation of different actinide oxidation states in solution. (author)

  1. Application of capillary electrophoresis to the simultaneous determination and stability study of four extensively used penicillin derivatives

    Directory of Open Access Journals (Sweden)

    Brigitta Simon

    2014-09-01

    Full Text Available The applicability of capillary electrophoresis for the analysis of four extensively used penicillin derivatives (benzylpenicillin, ampicillin, amoxicillin, oxacilllin has been studied. Because of structural similarities, the electrophoretic behavior of these derivatives is very similar; consequently an efficient separation using the conventional capillary zone electrophoresis is hard to be achieved. Their simultaneous separation was solved by using micellar electrokinetic capillary chromatography, the separation being based on the differential partition of the analytes between the micellar and aqueous phase. Using a buffer solution containing 25 mM sodium tetraborate and 100 mM sodium dodecyl sulfate as surfactant, at a pH of 9.3, applying a voltage of + 25 kV at a temperature of 25 °C, we achieved the simultaneous separation of the studied penicillin derivatives in less then 5 minutes. The separation conditions were optimized and the analytical performance of the method was evaluated in terms of precision, linearity, limit of detection, and quantification. Also, a simple capillary zone electrophoresis method was applied to study the stability of the studied penicillin derivatives in water at different temperatures, using ciprofloxacin hydrochloride as internal standard. It was observed that the extent of the hydrolysis of penicillins in water is highly dependent on the time and also temperature.

  2. 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)

  3. Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

    Science.gov (United States)

    Murphy, Sandra; Dowling, Paul; Ohlendieck, Kay

    2016-01-01

    The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins. PMID:28248237

  4. 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

  5. Electrophoresis of Ion Containing Polymers in Microfluidic Applications

    Science.gov (United States)

    Chow, Andrea

    2004-03-01

    Microfluidic technology offers the benefits of miniaturization, integration, and automation that can lead to faster analysis with higher data quality and higher sample throughput. One of the first microfluidic systems commercialized is for biomolecular sizing using the principles of gel electrophoresis. In these chips, the microchannels are filled with a polymer solution at a concentration above the entanglement threshold. For DNA and RNA sizing, the procedures of sample injection, fluorescent dye staining of the analyte, electrophoretic separation by size, and optical detection of size fractions are integrated. These microchip analyses are similar to those performed in conventional capillary electrophoresis, except that the analysis times are usually an order of magnitude shorter. For protein sizing in sodium dodecyl sulphate (SDS) micelle solutions in which the proteins are denatured, an additional step of protein destaining is also integrated onto the chip, enabling an application that has no simple analog in conventional capillary electrophoresis. The scaling laws based on polymer physics considerations dictating the sizing mechanisms and separation efficiencies for these microfluidic applications will be discussed.

  6. 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.

  7. Advances in capillary electrophoresis and the implications for drug discovery.

    Science.gov (United States)

    Ouimet, Claire M; D'amico, Cara I; Kennedy, Robert T

    2017-02-01

    Many screening platforms are prone to assay interferences that can be avoided by directly measuring the target or enzymatic product. Capillary electrophoresis (CE) and microchip electrophoresis (MCE) have been applied in a variety of formats to drug discovery. CE provides direct detection of the product allowing for the identification of some forms of assay interference. The high efficiency, rapid separations, and low volume requirements make CE amenable to drug discovery. Areas covered: This article describes advances in capillary electrophoresis throughput, sample introduction, and target assays as they pertain to drug discovery and screening. Instrumental advances discussed include integrated droplet microfluidics platforms and multiplexed arrays. Applications of CE to assays of diverse drug discovery targets, including enzymes and affinity interactions are also described. Expert opinion: Current screening with CE does not fully take advantage of the throughputs or low sample volumes possible with CE and is most suitable as a secondary screening method or for screens that are inaccessible with more common platforms. With further development, droplet microfluidics coupled to MCE could take advantage of the low sample requirements by performing assays on the nanoliter scale at high throughput.

  8. 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.

  9. 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.

  10. 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.)

  11. Determination of damage and In vivo DNA repairing through the unicellular in gel electrophoresis technique

    International Nuclear Information System (INIS)

    Mendiola C, M.T.; Morales R, P.

    1997-01-01

    The experimental conditions were standardized for the unicellular in gel electrophoresis technique setting up (EUG) at the Cellular Radiobiology laboratory. Preliminary experiments were realized with human cells and mouse which were exposed to ionizing radiation or hydroxide peroxide (H 2 O 2 ) to induce DNA damage and to verify the technique performance. It was analysed the In vivo repairing kinetics of induced damage by gamma radiation in mouse leukocytes which were exposed to 137 Cs source and taking samples of peripheric blood of the tail of each mouse at different exposure times and processing them for EUG. In function of the cells proportion with damage in each time it was determined the existence of fast repairing mechanism at the first 15 minutes followed by a slight increase in the damage and a late repairing stage between 30 and 90 minutes. It was analysed this behavior and the potentiality of this In vivo system. (Author)

  12. Calibration of low molecular weight polypeptides by sodium dodecylsulphate polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Glyn, M.C.P.; Bull, J.; Wright, R.

    1982-01-01

    Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique commonly used in determining molecular weights of large proteins and peptides. This technique is used to analyse viral peptides, available in amounts too small to be monitored by an ultraviolet spectrophotometer. An experiment is described (with the limiting factor to use the SDS-PAGE technique), to determine the molecular weight peptides and the results are given to fit the linear relationship log M=4.286 - 0.42 V(e)/V(o). The results given by the SDS-PAGE system, described in the article, show that the experimental values describe a linear relationship with good resolution of low molecular weight peptides in the range 3 000 to 14 000 and that a partial cyanogen bromide digest of cytochrome c is suitable for calibration standards

  13. [Establishment of two-dimensional differential gel electrophoresis using cerebrospinal fluid from neurocysticercosis patients].

    Science.gov (United States)

    Li, Jing-Yi; Tian, Xiao-Jun; Huang, Yong; Yang, Yan-Jun; Ma, Qiao-Rong; Xue, Yan-Ping

    2008-06-30

    To establish the method of two-dimensional differential gel electrophoresis and obtain high resolution 2D images from cerebrospinal fluid (CSF) of patients with neurocysticercosis. CSF samples were collected from four patients diagnosed as neurocysticercosis clinically and by ELISA, computed tomography (CT) or magnetic resonance imaging (MRI), and from four healthy subjects without neurological disorders. The CSF samples were precipitated with cold acetone, then pooled by equal amount as patients and controls. The internal standard comprised equal amounts of proteins extracted from both groups. Internal standard, and proteins from the two groups were labeled prior to electrophoresis with spectrally resolvable fluorescent dyes, cyanein dye2 (Cy2), Cy3 and Cy5. Sodium dodecylsulfonate polyacrylamide gel chromatography (SDS-PAGE) and two-dimensional differential in-gel electrophoresis (2-D DIGE) of labeled samples were then run. The differential expressed proteins showed in the images of SDS-PAGE and 2-D DIGE gels scanned with 488 nm, 532 nm and 633 nm wavelength laser were analyzed by ImageQuant and DeCyde 5.0 respectively. Spot detection and quantification was performed for the differential in-gel analysis (DIA) module of DeCyder. Biological variation analysis (BVA) module of DeCyder was matched gel 1 and gel 2 images to provide data on differential protein expression levels between the two groups. The ImageQuant result displayed that the CSF protein was compatible with the dye, and the difference of protein amount was revealed by the difference of fluorescence intensity. DIA indicated that there were 896 and 894 protein dots on gel 1 and gel 2 respectively, and 90% of them were matched each other. BVA showed that there were 55 protein spots with different expressional level between neurocysticercosis and control groups. Protein spots with two-fold increase or decrease were 47 and 8 respectively in neurocysticercosis patients compared with healthy controls. The

  14. Enhanced Agarose and Xylan Degradation for Production of Polyhydroxyalkanoates by Co-Culture of Marine Bacterium, Saccharophagus degradans and Its Contaminant, Bacillus cereus

    Directory of Open Access Journals (Sweden)

    Shailesh S. Sawant

    2017-02-01

    Full Text Available Over reliance on energy or petroleum products has raised concerns both in regards to the depletion of their associated natural resources as well as their increasing costs. Bioplastics derived from microbes are emerging as promising alternatives to fossil fuel derived petroleum plastics. The development of a simple and eco-friendly strategy for bioplastic production with high productivity and yield, which is produced in a cost effective manner utilising abundantly available renewable carbon sources, would have the potential to result in an inexhaustible global energy source. Here we report the biosynthesis of bioplastic polyhydroxyalkanoates (PHAs in pure cultures of marine bacterium, Saccharophagus degradans 2-40 (Sde 2-40, its contaminant, Bacillus cereus, and a co-culture of these bacteria (Sde 2-40 and B. cereus degrading plant and algae derived complex polysaccharides. Sde 2-40 degraded the complex polysaccharides agarose and xylan as sole carbon sources for biosynthesis of PHAs. The ability of Sde 2-40 to degrade agarose increased after co-culturing with B. cereus. The association of Sde 2-40 with B. cereus resulted in increased cell growth and higher PHA production (34.5% of dry cell weight from xylan as a carbon source in comparison to Sde 2-40 alone (22.7% of dry cell weight. The present study offers an innovative prototype for production of PHA through consolidated bioprocessing of complex carbon sources by pure and co-culture of microorganisms.

  15. Agarose-chitosan-C18film micro-solid phase extraction combined with high performance liquid chromatography for the determination of phenanthrene and pyrene in chrysanthemum tea samples.

    Science.gov (United States)

    Ng, Nyuk Ting; Sanagi, Mohd Marsin; Wan Ibrahim, Wan Nazihah; Wan Ibrahim, Wan Aini

    2017-05-01

    Agarose-chitosan-immobilized octadecylsilyl-silica (C 18 ) film micro-solid phase extraction (μSPE) was developed and applied for the determination of phenanthrene (PHE) and pyrene (PYR) in chrysanthemum tea samples using high performance liquid chromatography-ultraviolet detection (HPLC-UV). The film of blended agarose and chitosan allows good dispersion of C 18 , prevents the leaching of C 18 during application and enhances the film mechanical stability. Important μSPE parameters were optimized including amount of sorbent loading, extraction time, desorption solvent and desorption time. The matrix match calibration curves showed good linearity (r⩾0.994) over a concentration range of 1-500ppb. Under the optimized conditions, the proposed method showed good limits of detection (0.549-0.673ppb), good analyte recoveries (100.8-105.99%) and good reproducibilities (RSDs⩽13.53%, n=3) with preconcentration factors of 4 and 72 for PHE and PYR, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Development of a chamber system for rapid, high yield and cost-effective purification of deoxyribonucleic acid fragments from agarose gel.

    Science.gov (United States)

    Eslami, Gilda; Salehi, Rasoul

    2014-01-01

    There are several methods commonly practicing for deoxyribonucleic acid (DNA) purification from agarose gel. In most laboratories, especially in developing countries, present methods for recovering of DNA fragments from the gel are mostly involved organic solvents. However, manual purification using organic solvents are toxic, labor intensive, time consuming and prone to contamination owing to several handling steps. The above mentioned burdens as well as cost and long time to import them, especially in developing countries, prompted us to design and develop a chamber system for rapid, non-toxic, cost-effective and user friendly device for polymerase chain reaction (PCR) products purification from agarose gel. The device was made from plexiglass plates. After amplification of two fragments of 250 and 850 bp, PCR products were electrophoresed. Subsequently, the desired bands were excised and purified with three method: HiPer Mini chamber, phenol extraction method and spin column procedure. To assess the suitability of the purified DNAs, restriction digestion was applied. Results showed that the yield of recovered DNA in our method was above 95%, whereas the yields obtained with conventional phenol extraction and spin column methods were around 60%. In conclusion, the current method for DNA elution is quick, inexpensive and robust and it does not require the use of toxic organic solvents. In addition, the purified DNA was well has suited for further manipulations such as restriction digestion, ligation, cloning, sequencing and hybridization.

  17. Co-immobilization of cyclohexanone monooxygenase and glucose-6-phosphate dehydrogenase onto polyethylenimine-porous agarose polymeric composite using γ irradiation to use in biotechnological processes

    International Nuclear Information System (INIS)

    Atia, K.S.

    2005-01-01

    The co-immobilization of cyclohexanone monooxygenase (CHMO) and glucose-6-phosphate dehydrogenase (G6PDH) was optimized by completely coating, via covalent immobilization, the surface aldehyde groups of porous agarose (glyoxyl-agarose) with amine groups of polyethylenimine (PEI). The highest immobilization efficiency (∼87%) (activity of enzyme per amount of immobilized enzyme) was obtained with a CHMO/G6PDH ratio 2:1. The effects of different ratios of the support to the amount of enzymes (CHMO:G6PDH=2:1), the optimum incubation pH and the incubation time on the enzymatic activity of the enzymes were determined and found to be 5:1, 8.5 and 30 min, respectively. Subjecting the co-immobilized enzymes to doses of γ-radiation (5-100 kGy) resulted in complete loss in the activity of the free enzymes at a dose of 40 kGy, while the co-immobilized ones showed relatively high resistance to γ-radiation up to a dose of 50 kGy

  18. Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Petersen, Jesper; Stoltenborg, M.

    2006-01-01

    Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an ag......Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation...... to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose Mutations in the human P-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding inciting point temperature (similar to 20...... degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments...

  19. 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.

  20. 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)

  1. 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

  2. Quantitative gel electrophoresis: new records in precision by elaborated staining and detection protocols.

    Science.gov (United States)

    Deng, Xi; Schröder, Simone; Redweik, Sabine; Wätzig, Hermann

    2011-06-01

    Gel electrophoresis (GE) is a very common analytical technique for proteome research and protein analysis. Despite being developed decades ago, there is still a considerable need to improve its precision. Using the fluorescence of Colloidal Coomassie Blue -stained proteins in near-infrared (NIR), the major error source caused by the unpredictable background staining is strongly reduced. This result was generalized for various types of detectors. Since GE is a multi-step procedure, standardization of every single step is required. After detailed analysis of all steps, the staining and destaining were identified as the major source of the remaining variation. By employing standardized protocols, pooled percent relative standard deviations of 1.2-3.1% for band intensities were achieved for one-dimensional separations in repetitive experiments. The analysis of variance suggests that the same batch of staining solution should be used for gels of one experimental series to minimize day-to-day variation and to obtain high precision. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. 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)

  4. Electrophoresis in charge-stabilized colloidal cluster phases.

    Science.gov (United States)

    Groenewold, Jan; Zhang, Tianhui; Kegel, Willem K

    2011-06-09

    The reversible properties of cluster phases have been described by theories that invoke Coulomb interactions as a stabilizing mechanism. What is lacking so far is direct measurement of these charges. This contribution aims at predicting what to expect if electrophoresis measurements were to be performed on these systems. As a result, we get a picture that exhibits several interesting features: (1) The existence of monomers and clusters lead to distinctly different mobilities (zeta potentials) in a single sample. (2) Strong dependence of the mobilities on particle volume fraction. It is our aim that the theory outlined in this paper may serve as a guideline to interpret the expectedly "messy" electrophoretic measurements.

  5. Genotyping of Ureaplasma diversum isolates using pulsed-field electrophoresis.

    Science.gov (United States)

    Buzinhani, Melissa; Buim, Marcos R; Yamaguti, Maurício; Oliveira, Rosângela C; Mettifogo, Elena; Timenetsky, Jorge

    2007-05-01

    Ureaplasma diversum has been associated with reproductive disorders in cattle and in the present study genotypic variations among U. diversum isolates obtained from the vaginal mucus of healthy cattle and sick animals were analyzed by enzymatic digestion and pulsed-field gel electrophoresis (PFGE). The influence of time and broth volume was important in obtaining sufficient cell sediment and DNA for PFGE. The method presented a high discriminatory power and satisfactory reproducibility for the analysis of detected variations among U. diversum isolates and strains. Different band profiles and wide genotypic heterogeneity were detected but no association between DNA polymorphism and sick or healthy animals could be established.

  6. Comparative gel electrophoresis analysis of helical junctions in RNA.

    Science.gov (United States)

    Lilley, David M J

    2009-01-01

    Comparative gel electrophoresis provides information on the relative angles subtended between helical arms at a branchpoint in RNA. It is based upon the comparison of electrophoretic mobility in polyacrylamide gels of species containing two long arms, with the remaining one(s) being significantly shorter. Although the method currently lacks a really well-established basis of physical theory, it is very powerful, yet simple to apply. It has had a number of significant successes in RNA, DNA and DNA-protein complexes, and in all cases to date the results have stood the test of time and eventual comparison with crystallographic analysis. Copyright © 2009 Elsevier Inc. All rights reserved.

  7. Polyacrylamide Gel Electrophoresis for Purification of Large Amounts of RNA.

    Science.gov (United States)

    Meyer, Mélanie; Masquida, Benoît

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) constitutes a powerful technique for the efficient purification of RNA molecules dedicated to applications that require high purity levels. PAGE allows for the fractionation of RNA obtained from cell extracts, chemical or enzymatic synthesis, or modification experiments. Native or denaturing conditions can be chosen for analytical or preparative-scale separations and the nucleotide resolution can be tuned by changing the percentage and reticulation of the gel material. In this protocol, we focus on the preparation of milligram-scale amounts of ~200 nucleotides (nt) RNA molecules that were used in subsequent crystallization experiments.

  8. Analysis of RNA folding by native polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Woodson, Sarah A; Koculi, Eda

    2009-01-01

    Polyacrylamide gel electrophoresis under native conditions (native PAGE) is a well-established and versatile method for probing nucleic acid conformation and nucleic acid-protein interactions. Native PAGE has been used to measure RNA folding equilibria and kinetics under a wide variety of conditions. Advantages of this method are its adaptability, absolute determination of reaction endpoints, and direct analysis of conformational hetereogeneity within a sample. Native PAGE is also useful for resolving ligand-induced structural changes. Copyright © 2009 Elsevier Inc. All rights reserved.

  9. DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis

    Science.gov (United States)

    Wang, Hui; Dunning, James E.; Huang, Albert P.-H.; Nyamwanda, Jacqueline A.; Branton, Daniel

    2004-09-01

    Broad-spectrum analysis of DNA and RNA samples is of increasing importance in the growing field of biotechnology. We show that nanopore measurements may be used to assess the purity, phosphorylation state, and chemical integrity of nucleic acid preparations. In contrast with gel electrophoresis and mass spectrometry, an unprecedented dynamic range of DNA sizes and concentrations can be evaluated in a single data acquisition process that spans minutes. Because the molecule information is quantized and digitally recorded with single-molecule resolution, the sensitivity of the system can be adjusted in real time to detect trace amounts of a particular DNA species.

  10. 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.

  11. 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)

  12. 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

  13. 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

  14. Developments and Applications of Electrophoresis and Small Molecule Laser Desorption Ionization Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hui [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    Ultra-sensitive native fluorescence detection of proteins with miniaturized one- and two-dimensional polyacrylamide gel electrophoresis was achieved with laser side-entry excitation, which provides both high excitation power and low background level. The detection limit for R-phycoerythrin protein spots in 1-D SDS-PAGE was as low as 15 fg, which corresponds to 40 thousand molecules only. The average detection limit of six standard native proteins was 5 pg per band and the dynamic range spanned more than 3 orders of magnitude. Approximately 150 protein spots from 30 ng of total Escherichia coli extraction were detected on a 0.8 cm x 1 cm gel in two-dimensional separation. Estrogen-DNA adducts as 4-OHE1(E2)-1-N3Ade and 4-OHEI(E2)-2-NacCys were hypothesized as early risk assessment of prostate and breast cancers. Capillary electrophoresis, luminescence/absorption spectroscopy and LC-MS were used to characterize and detect these adducts. Monoclonal antibodies against each individual adduct were developed and used to enrich such compounds from urine samples of prostate and breast cancer patients as well as healthy people. Adduct 4-OHE1-1-N3Ade was detected at much higher level in urine from subjects with prostate cancer patients compared to healthy males. The same adduct and 4-OHEI-2-NacCys were also detected at a much higher level in urine from a woman with breast carcinoma than samples from healthy controls. These two DNA adducts may serve as novel biomarkers for early diagnostic of cancers. The adsorption properties of R-phycoerythrin (RPE), on the fused-silica surface were studied using capillary electrophoresis (CE) and single molecule spectroscopy. The band shapes and migration times were measured in CE. Adsorption and desorption events were recorded at the single-molecule level by imaging of the evanescent-field layer using total internal reflection. The adsorbed RPE molecules on the fused-silica prism surface were

  15. Fast high-throughput method for the determination of acidity constants by capillary electrophoresis: I. Monoprotic weak acids and bases.

    Science.gov (United States)

    Fuguet, Elisabet; Ràfols, Clara; Bosch, Elisabeth; Rosés, Martí

    2009-04-24

    A new and fast method to determine acidity constants of monoprotic weak acids and bases by capillary zone electrophoresis based on the use of an internal standard (compound of similar nature and acidity constant as the analyte) has been developed. This method requires only two electrophoretic runs for the determination of an acidity constant: a first one at a pH where both analyte and internal standard are totally ionized, and a second one at another pH where both are partially ionized. Furthermore, the method is not pH dependent, so an accurate measure of the pH of the buffer solutions is not needed. The acidity constants of several phenols and amines have been measured using internal standards of known pK(a), obtaining a mean deviation of 0.05 pH units compared to the literature values.

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

    Directory of Open Access Journals (Sweden)

    Jae Eun Lee

    2015-06-01

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

  17. Automated band annotation for RNA structure probing experiments with numerous capillary electrophoresis profiles.

    Science.gov (United States)

    Lee, Seungmyung; Kim, Hanjoo; Tian, Siqi; Lee, Taehoon; Yoon, Sungroh; Das, Rhiju

    2015-09-01

    Capillary electrophoresis (CE) is a powerful approach for structural analysis of nucleic acids, with recent high-throughput variants enabling three-dimensional RNA modeling and the discovery of new rules for RNA structure design. Among the steps composing CE analysis, the process of finding each band in an electrophoretic trace and mapping it to a position in the nucleic acid sequence has required significant manual inspection and remains the most time-consuming and error-prone step. The few available tools seeking to automate this band annotation have achieved limited accuracy and have not taken advantage of information across dozens of profiles routinely acquired in high-throughput measurements. We present a dynamic-programming-based approach to automate band annotation for high-throughput capillary electrophoresis. The approach is uniquely able to define and optimize a robust target function that takes into account multiple CE profiles (sequencing ladders, different chemical probes, different mutants) collected for the RNA. Over a large benchmark of multi-profile datasets for biological RNAs and designed RNAs from the EteRNA project, the method outperforms prior tools (QuSHAPE and FAST) significantly in terms of accuracy compared with gold-standard manual annotations. The amount of computation required is reasonable at a few seconds per dataset. We also introduce an 'E-score' metric to automatically assess the reliability of the band annotation and show it to be practically useful in flagging uncertainties in band annotation for further inspection. The implementation of the proposed algorithm is included in the HiTRACE software, freely available as an online server and for download at http://hitrace.stanford.edu. sryoon@snu.ac.kr or rhiju@stanford.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Boronate affinity electrophoresis for the purification and analysis of cofactor-modified RNAs.

    Science.gov (United States)

    Nübel, Gabriele; Sorgenfrei, Frieda A; Jäschke, Andres

    2017-03-15

    RNA modifications are widely distributed in Nature, and their thorough analysis helps answering fundamental biological questions. Nowadays, mass spectrometry or deep-sequencing methods are often used for the analysis. With the raising number of newly discovered RNA modifications, such as the 5'-NAD cap in Escherichia coli, there is an important need for new, less complex and fast analytical tools to analyze the occurrence, amount, and distribution of modified RNAs in cells. To accomplish this task, we have revisited the previously developed affinity gel electrophoresis principles and copolymerized acryloylaminophenyl boronic acid (APB) in standard denaturing polyacrylamide gels to retard the NAD- or FAD-modified RNAs compared to the unmodified RNAs in the gels. The boronyl groups inside the gel form relatively stable complexes with 1,2-cis diols, occurring naturally at the 3'-end of RNA, and also in the nicotinamide riboside of NAD-modified RNA at the 5'-end. The transient formation of diesters between the immobilized boronic acid and the diols causes lower mobility of the modified RNAs, compared to unmodified RNAs, resulting in two distinct bands for one RNA sequence. We used APB affinity gel electrophoresis to preparatively purify in vitro transcribed NAD-RNA from triphosphorylated RNA, to study the enzyme kinetics of the NAD-RNA decapping enzyme NudC, and to determine the NAD modification ratios of various cellular sRNAs. In summary, APB affinity gels can be used to study cofactor-modified RNAs with low amounts of material, and to rapidly screen for their occurrence in total RNA while avoiding complex sample treatments. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. [Study on the method of two dimensional polycrylamide gel electrophoresis on rat condylar chondrocyte].

    Science.gov (United States)

    Wu, Tuo-jiang; Li, Huang; Ma, Qiao-lin; Wang, Wen-mei

    2010-08-01

    To investigate the protein profile by two dimensional polycrylamide gel electrophoresis on the rat condylar chondrocyte in vitro. The third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats in this study. The protein profile of the rat mandibular condylar chondrocytes was examined by two dimensional polycrylamide gel electrophoresis (2-DE-PAGE). The 2-DE gel maps on different pH gradients were obtained. The result of modified coomassi blue-sliver staining and sliver staining was compared using Pdquest 7.1 image analysis software. The results showed that the good protein profile of the condylar chondrocytes was obtained by standard Bio-Rad manual. The protein was mainly in the field from pH4 to pH7. The 1203±86 protein points were examined on 2-DE gel map by modified coomassi blue-sliver staining, and 1769±97 protein points was examined by sliver staining. The silver staining map showed more distinctly but higher background than modified coomassi blue-sliver staining. The protein profile of the condylar chondrocytes enriches the proteomic database and gives evidence to further proteomic research. The 2-DE map obtained by modified coomassi blue-sliver staining is more suitable for MALDI-TOF mass identification. Supported by National Natural Science Foundation of China (Grant No. C30700963), China Postdoctoral Science Foundation(Grant No.20090461088), Jiangsu Provincial Postdoctoral Science Foundation (Grant No.0802003C) and Nanjing City's Science and Technology Foundation (Grant No.200905011).

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

    Science.gov (United States)

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

    2015-01-01

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

  1. Polysaccharides of algae. Pt. 37. Characterization of hybrid structure of substituted agarose from Polysiphonia morrowii (Rhodophyta, Rhodomelaceae) using. beta. -agarase and /sup 13/C-NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Usov, A.I.; Ivanova, E.G.

    1987-09-01

    Structure of gel-forming galactan from Polysiphonia morrowii was analysed using bacterial ..beta..-agarase and /sup 13/C-nuclear magnetic resonance (/sup 13/C-NMR) spectroscopy. The polysaccharide was shown to contain: a) blocks composed of agarobiose residues, partly 6-O-methylated and 6-sulfated, which are sensitive to enzymolysis; b) extended blocks composed of agarobiose 6-sulfate residues, which are resistant to ..beta..-agarase action. The latter blocks contain also ..beta..-D-galactopyranosyl-(1->4)-..cap alpha..-L-galactopyranose 6.6'-disulfate residues (biogenetic precursors of agarobiose 6-sulfate), which are hardly detectable by /sup 13/C-NMR spectrum of the starting polysaccharide. Action of alkali on the enzyme-resistant fraction afforded a polysaccharide preparation having /sup 13/C-NMR spectrum of agarose 6-sulfate.

  2. The effects of cyclic hydrostatic pressure on chondrogenesis and viability of human adipose- and bone marrow-derived mesenchymal stem cells in three-dimensional agarose constructs.

    Science.gov (United States)

    Puetzer, Jennifer; Williams, John; Gillies, Allison; Bernacki, Susan; Loboa, Elizabeth G

    2013-01-01

    This study investigates the effects of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3-D) agarose constructs maintained in a complete growth medium without soluble chondrogenic inducing factors. hASCs were seeded in 2% agarose hydrogels and exposed to 7.5 MPa CHP for 4 h per day at a frequency of 1 Hz for up to 21 days. On days 0, 7, 14, and 21, the expression levels of collagen II, Sox9, aggrecan, and cartilage oligomeric matrix protein (COMP) were examined by real-time reverse transcriptase-polymerase chain reaction analysis. Gene expression analysis found collagen II mRNA expression in only the CHP-loaded construct at day 14 and at no other time during the study. CHP-loaded hASCs exhibited upregulated mRNA expression of Sox9, aggrecan, and COMP at day 7 relative to unloaded controls, suggesting that CHP initiated chondrogenic differentiation of hASCs in a manner similar to human bone marrow-derived mesenchymal stem cells (hMSC). By day 14, however, loaded hASC constructs exhibited significantly lower mRNA expression of the chondrogenic markers than unloaded controls. Additionally, by day 21, the samples exhibited little measurable mRNA expression at all, suggesting a decreased viability. Histological analysis validated the lack of mRNA expression at day 21 for both the loaded and unloaded control samples with a visible decrease in the cell number and change in morphology. A comparative study with hASCs and hMSCs further examined long-term cell viability in 3-D agarose constructs of both cell types. Decreased cell metabolic activity was observed throughout the 21-day experimental period in both the CHP-loaded and control constructs of both hMSCs and hASCs, suggesting a decrease in cell metabolic activity, alluding to a decrease in cell viability. This suggests that a 2% agarose hydrogel may not optimally support hASC or hMSC viability in a complete growth medium in the

  3. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hao-Tsai Cheng

    2016-01-01

    Full Text Available Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established. Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE profiling quality, including number of protein spots and spot distribution. Results. The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin. Conclusions. Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images.

  4. Disposable polyester-toner electrophoresis microchips for DNA analysis.

    Science.gov (United States)

    Duarte, Gabriela R M; Coltro, Wendell K T; Borba, Juliane C; Price, Carol W; Landers, James P; Carrilho, Emanuel

    2012-06-07

    Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215,000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.

  5. Analysis of RNA by analytical polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Petrov, Alexey; Tsa, Albet; Puglisi, Joseph D

    2013-01-01

    Polyacrylamide gel electrophoresis (PAGE) is a powerful tool for analyzing RNA samples. Denaturing PAGE provides information on the sample composition and structural integrity of the individual RNA species. Nondenaturing gel electrophoresis allows separation of the conformers and alternatively folded RNA species. It also can be used to resolve RNA protein complexes and to detect RNA complex formation by analyzing changes in the electrophoretic mobility of the RNA. RNA can be visualized within gels by different methods depending on the nature of the detection reagent. RNA molecules can be stained with various dyes, including toluidine blue, SYBR green, and ethidium bromide. Radioactively labeled RNA molecules are visualized by autoradiography, and fluorescently labeled RNA molecules can be observed with a fluorescence scanner. Generally, gels between 0.4 and 1.5mm thick are used for analytical PAGE. Gels thinner than 1mm are fragile and thus usually are not stained but rather are used for radiolabeled RNA. The gels are dried and the radiolabeled RNA is visualized by autoradiography. © 2013 Elsevier Inc. All rights reserved.

  6. Startup of electrophoresis in a suspension of colloidal spheres.

    Science.gov (United States)

    Chiang, Chia C; Keh, Huan J

    2015-12-01

    The transient electrophoretic response of a homogeneous suspension of spherical particles to the step application of an electric field is analyzed. The electric double layer encompassing each particle is assumed to be thin but finite, and the effect of dynamic electroosmosis within it is incorporated. The momentum equation for the fluid outside the double layers is solved through the use of a unit cell model. Closed-form formulas for the time-evolving electrophoretic and settling velocities of the particles in the Laplace transform are obtained in terms of the electrokinetic radius, relative mass density, and volume fraction of the particles. The time scale for the development of electrophoresis and sedimentation is significantly smaller for a suspension with a higher particle volume fraction or a smaller particle-to-fluid density ratio, and the electrophoretic mobility at any instant increases with an increase in the electrokinetic particle radius. The transient electrophoretic mobility is a decreasing function of the particle volume fraction if the particle-to-fluid density ratio is relatively small, but it may increase with an increase in the particle volume fraction if this density ratio is relatively large. The particle interaction effect in a suspension on the transient electrophoresis is much weaker than that on the transient sedimentation of the particles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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.

  8. 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).

  9. Two-Dimensional Gel Electrophoresis and 2D-DIGE.

    Science.gov (United States)

    Meleady, Paula

    2018-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to be one of the most versatile and widely used techniques to study the proteome of a biological system. In particular, a modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labeling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE gels for differential quantitative analysis of protein expression between experimental groups. Both these methods have distinct advantages in the separation and identification of thousands of individual proteins species including protein isoforms and post-translational modifications. This review will discuss the principles of 2D-PAGE and 2D-DIGE including limitations to the methods. 2D-PAGE and 2D-DIGE continue to be popular methods in bioprocessing-related research (particularly on recombinant Chinese hamster ovary cells), which will also be discussed in the review chapter.

  10. 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.

  11. Vibrio algivorus sp. nov., an alginate- and agarose-assimilating bacterium isolated from the gut flora of a turban shell marine snail.

    Science.gov (United States)

    Doi, Hidetaka; Chinen, Akito; Fukuda, Hiroo; Usuda, Yoshihiro

    2016-08-01

    An agarose- and alginate-assimilating, Gram-reaction-negative, non-motile, rod-shaped bacterium, designated strain SA2T, was isolated from the gut of a turban shell sea snail (Turbo cornutus) collected near Noto Peninsula, Ishikawa Prefecture, Japan. The 16S rRNA gene sequence of strain SA2T was 99.59 % identical to that of Vibrio rumoiensis DSM 19141T and 98.19 % identical to that of Vibrio litoralis DSM 17657T. This suggested that strain SA2T could be a subspecies of V. rumoiensis or V. litoralis. However, DNA-DNA hybridization results showed only 37.5 % relatedness to DSM 19141T and 44.7 % relatedness to DSM 17657T, which was far lower than the 70 % widely accepted to define common species. Strain SA2T could assimilate agarose as a sole carbon source, whereas strains DSM 19141T and DSM 17657T could not assimilate it at all. Furthermore, results using API 20NE and API ZYM kits indicated that their enzymic and physiological phenotypes were also different. These results suggested that strain SA2T represented a novel species within the genus Vibrio. The major isoprenoid quinone in SA2T was Q-8, and its major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major fatty acids were summed feature 3, (comprising C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0, and summed feature 8 (comprising C18 : 1ω6c and/or C18 : 1ω7c). The DNA G+C content of SA2T was 40.7 mol%. The name proposed for this novel species of the genus Vibrio is Vibrio algivorus sp. nov., with the type strain designated SA2T (=DSM 29824T=NBRC 111146T).

  12. Analysis of the genome of a Korean isolate of the Pieris rapae granulovirus enabled by its separation from total host genomic DNA by pulse-field electrophoresis.

    Directory of Open Access Journals (Sweden)

    Yong Hun Jo

    Full Text Available BACKGROUND: Most traditional genome sequencing projects involving viruses include the culture and purification of the virus particles. However, purification of virions may yield insufficient material for traditional sequencing. The electrophoretic method described here provides a strategy whereby the genomic DNA of the Korean isolate of Pieris rapae granulovirus (PiraGV-K could be recovered in sufficient amounts for sequencing by purifying it directly from total host DNA by pulse-field gel electrophoresis (PFGE. METHODOLOGY/PRINCIPAL FINDINGS: The total genomic DNA of infected P. rapae was embedded in agarose plugs, treated with restriction nuclease and methylase, and then PFGE was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the purified viral DNA. The double-stranded circular genome of PiraGV-K was found to encode 120 open reading frames (ORFs, which covered 92% of the sequence. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%, followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10 and cathepsin (ORF 11, involved in the liquefaction of the host, were found in the genome. CONCLUSIONS/SIGNIFICANCE: The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the electrophoretic method. The method appears to be generally applicable to the analysis of genomes of large viruses.

  13. Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence.

    Science.gov (United States)

    García-Cañas, Virginia; González, Ramón; Cifuentes, Alejandro

    2004-07-01

    The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5-0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge

  14. Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification.

    Science.gov (United States)

    Liu, Peng; Li, Xiujun; Greenspoon, Susan A; Scherer, James R; Mathies, Richard A

    2011-03-21

    A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification. This journal is © The Royal Society of Chemistry 2011

  15. A Direct and Rapid Method to Determine Cyanide in Urine by Capillary Electrophoresis

    Science.gov (United States)

    Zhang, Qiyang; Maddukuri, Naveen; Gong, Maojun

    2015-01-01

    Cyanides are poisonous chemicals that widely exist in nature and industrial processes as well as accidental fires. Rapid and accurate determination of cyanide exposure would facilitate forensic investigation, medical diagnosis, and chronic cyanide monitoring. Here, a rapid and direct method was developed for the determination of cyanide ions in urinary samples. This technique was based on an integrated capillary electrophoresis system coupled with laser-induced fluorescence (LIF) detection. Cyanide ions were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and a primary amine (glycine) for LIF detection. Three separate reagents, NDA, glycine, and cyanide sample, were mixed online, which secured uniform conditions between samples for cyanide derivatization and reduced the risk of precipitation formation of mixtures. Conditions were optimized; the derivatization was completed in 2-4 minutes, and the separation was observed in 25 s. The limit of detection (LOD) was 4.0 nM at 3-fold signal-to-noise ratio for standard cyanide in buffer. The cyanide levels in urine samples from smokers and non-smokers were determined by using the method of standard addition, which demonstrated significant difference of cyanide levels in urinary samples from the two groups of people. The developed method was rapid and accurate, and is anticipated to be applicable to cyanide detection in waste water with appropriate modification. PMID:26342870

  16. Serum biochemistry and hematology values and hemoglobin electrophoresis in Persian squirrels (Sciurus anomalus).

    Science.gov (United States)

    Asadi, Farzad; Rostami, Amir; Asadian, Peyman; Pourkabir, Malihe

    2007-06-01

    Serum biochemical and hematologic parameters are important in the management of game species in Iran, such as Persian squirrels. The purpose of this study was to establish baseline serum chemistry and hematology values in Persian squirrels (Sciurus anomalus) and describe blood cell morphology and the electrophoretic pattern of hemoglobin. Blood samples were collected from 30 Persian squirrels (Sciurus anomalus) maintained in captivity in the Tehran Zoo. Blood was placed into EDTA and serum clot tubes and analyzed using standard manual hematology and biochemical techniques. Hemoglobin electrophoresis was done on cellulose acetate paper strips. Minimum, maximum, and median values were obtained for 11 hematologic and 12 serum chemistry parameters. Hypersegmented neutrophils were observed frequently. We did not find basophils or band neutrophils. Hemoglobin electophoresis resulted in a band slightly anodal to that of human hemoglobin A. Biochemical and hematologic values in Persian squirrels were comparable to those of related species, and may be used as a standard profile for healthy Persian squirrels kept in captivity.

  17. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

    Science.gov (United States)

    Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

    2016-09-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

  18. Heat stress-induced loss of eukaryotic initiation factor 5A (eIF-5A) in a human pancreatic cancer cell line, MIA PaCa-2, analyzed by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Takeuchi, Kana; Nakamura, Kazuyuki; Fujimoto, Masanori; Kaino, Seiji; Kondoh, Satoshi; Okita, Kiwamu

    2002-02-01

    Alterations of intracellular proteins during the process of heat stress-induced cell death of a human pancreatic cancer cell line, MIA PaCa-2, were investigated using two-dimensional gel electrophoresis (2-DE), agarose gel electrophoresis, and cell biology techniques. Incubation of MIA PaCa-2 at 45 degrees C for 30 min decreased the cell growth rate and cell viability without causing chromosomal DNA fragmentation. Incubation at 51 degrees C for 30 min suppressed cell growth and again led to death without DNA fragmentation. The cell death was associated with the loss of an intracellular protein of M(r) 17,500 and pI 5.2 on 2-DE gel. This protein was determined to be eukaryotic initiation factor SA (eIF-5A) by microsequencing of the N-terminal region of peptide fragments obtained by cyanogen bromide treatment of the protein blotted onto a polyvinylidene difluoride (PVDF) membrane. The sequences detected were QXSALRKNGFVVLKGRP and STSKTGXHGHAKVHLVGID, which were homologous with the sequence of eIF-5A from Gln 20 to Pro 36 and from Ser 43 to Asp 61, respectively. Furthermore, the result of sequencing suggested that the protein was an active form of hypusinated eIF-5A, because Lys 46 could be detected but not Lys 49, which is the site for hypusination. These results suggest that loss of the active form of eIF-5A is an important factor in the irreversible process of heat stress-induced death of MIA PaCa-2 cells.

  19. Analysis of lipoproteins by capillary zone electrophoresis in microfluidic devices: Assay development and surface roughness measurements

    NARCIS (Netherlands)

    Weiller, Bruce H.; Ceriotti, Laura; Shibata, Takayuki; Rein, Dietrich; Roberts, Matthew A.; Lichtenberg, Jan; German, J. Bruce; De Rooij, Nico F.; Verpoorte, Elisabeth

    2002-01-01

    The development of a new assay for lipoproteins by capillary electrophoresis in fused-silica capillaries and in glass microdevices is described in this paper. The separation of low-density (LDL) and high-density (HDL) lipoproteins by capillary zone electrophoresis is demonstrated in fused-silica

  20. Instrumental development of novel detection and separation methods for capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Garner, Tommy [Iowa State Univ., Ames, IA (United States)

    1993-07-01

    After a general introduction, this thesis is divided into 3 parts: indirect fluorescence detection of sugars separated by capillary zone electrophoresis with visible laser excitation, absorption detection in capillary electrophoresis by fluorescence energy transfer, and increased selectivity for electrochromatography by dynamic ion exchange.

  1. Simulation of Two Dimensional Electrophoresis and Tandem Mass Spectrometry for Teaching Proteomics

    Science.gov (United States)

    Fisher, Amanda; Sekera, Emily; Payne, Jill; Craig, Paul

    2012-01-01

    In proteomics, complex mixtures of proteins are separated (usually by chromatography or electrophoresis) and identified by mass spectrometry. We have created 2DE Tandem MS, a computer program designed for use in the biochemistry, proteomics, or bioinformatics classroom. It contains two simulations--2D electrophoresis and tandem mass spectrometry.…

  2. Identification of ectoproteins of human platelets. Combination of radioactive labelling and two-dimensional electrophoresis

    NARCIS (Netherlands)

    Sixma, J.J.; Schiphorst, M.E.

    1980-01-01

    Two-dimensional gel electrophoresis combining isoelectric focussing of reduced or non-reduced proteins in the first dimension with electrophoresis in sodium dodecyl sulfate polyacrylamide gels in the second dimension enabled us to identify 25 ectoproteins in the non-reduced state and 32 in the

  3. 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...... and pharmaceutical applications, e.g., drug development. The technique results in an image, where the proteins appear as dark spots on a bright background. However, the analysis of these images is very time consuming and requires a large amount of manual work so there is a great need for fast, objective, and robust...... methods based on image analysis techniques in order to significantly accelerate this key technology. The methods described and developed fall into three categories: image segmentation, point pattern matching, and a unified approach simultaneously segmentation the image and matching the spots. The main...

  4. Preparation of MgB2 superconducting tapes using electrophoresis

    Science.gov (United States)

    Xu, J. D.; Wang, S. F.; Zhou, Y. B.; Zhou, Y. L.; Chen, Z. H.; Cui, D. F.; Lu, H. B.; He, M.; Dai, S. Y.; Yang, G. Z.

    2002-08-01

    Superconducting MgB2/Ta tapes with a critical temperature of 34 K have been prepared successfully by ex situ annealing of electrophoresis-grown boron in the presence of Mg vapour at 920 °C. Scanning electron microscopy was used to examine the surface morphology of the MgB2/Ta tapes, and well-formed MgB2 crystals with sizes up to 2 μm were observed. The x-ray diffraction patterns showed randomly orientated growth of MgB2 phase in the tapes. Estimates using hysteresis loops and the Bean model give a value of 6.8 × 105 A cm-2 for the critical current density.

  5. Determination of organic contaminants in food by capillary electrophoresis.

    Science.gov (United States)

    Juan-García, Ana; Font, Guillermina; Picó, Yolanda

    2005-06-01

    This review addresses recent advances in the analysis of organic contaminants, such as antibiotics, pesticides, biological toxins, and food-borne pathogens, in foods by capillary electrophoresis (CE). Special attention is paid to those aspects that increase sensitivity and/or selectivity, such as sample extraction and concentration, on-line preconcentration techniques (stacking), affinity capillaries or/and specific detectors (laser induced fluorescence (LIF), mass spectrometry (MS)). The various CE modes used to separate the compounds and the quantification strategies are also examined. As a result, this work presents an updated overview on the principal applications of CE, together with a discussion of their main advantages and drawbacks, and an outline of future trends in the analysis of organic contaminants in food.

  6. Sample preparation guidelines for two-dimensional electrophoresis.

    Science.gov (United States)

    Posch, Anton

    2014-12-01

    Sample preparation is one of the key technologies for successful two-dimensional electrophoresis (2DE). Due to the great diversity of protein sample types and sources, no single sample preparation method works with all proteins; for any sample the optimum procedure must be determined empirically. This review is meant to provide a broad overview of the most important principles in sample preparation in order to avoid a multitude of possible pitfalls. Sample preparation protocols from the expert in the field were screened and evaluated. On the basis of these protocols and my own comprehensive practical experience important guidelines are given in this review. The presented guidelines will facilitate straightforward protocol development for researchers new to gel-based proteomics. In addition the available choices are rationalized in order to successfully prepare a protein sample for 2DE separations. The strategies described here are not limited to 2DE and can also be applied to other protein separation techniques.

  7. Determination of dyes in foodstuffs by capillary zone electrophoresis.

    Science.gov (United States)

    Pérez-Urquiza, M; Beltrán, J L

    2000-11-17

    A rapid method based on capillary zone electrophoresis coupled with photodiode-array detection has been developed to determine the dyes Tartrazine E-102, Sunset Yellow FCF E110, Amaranth E-123, New Coccine E-124, Patent Blue V calcium salt E-131 and Allura Red AC E-129 in foodstuffs. Separation was done by using a Bare CElect-FS75 CE column, using a 10 mM phosphate buffer at pH 11.0. Hydrodynamic injections at 0.5 p.s.i. for 4 s (21 nl of sample) and 20 kV separation voltage were used. The quantitation limits for the six dyes ranged from 3 to 6 microg/ml. A linear relationship between 3 to 95 microg/ml, with correlation coefficient better than 0.995 was obtained. This method has been applied to the determination of the studied dyes in beverages, jellies and syrups.

  8. 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...... determined the x-ray crystal structures of both plant and human glucan phosphatases and their enzymatic mechanisms. Despite this progress, we lacked the techniques to quickly and efficiently quantify their glucan phosphatase affinities for different substrates. The main objective of this study...... and animal glucan phosphatases to determine which regions of the enzyme are most necessary for binding. Footnotes This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal....

  9. Identification of RNA-ligand interactions by affinity electrophoresis.

    Science.gov (United States)

    Boodram, Sherry N; Cho, Chul M; Tavares, Tony J; Johnson, Philip E

    2011-02-01

    We have developed an affinity electrophoresis method to screen for RNA-ligand interactions. Native polyacrylamide gels were polymerized in the absence and presence of different RNA binding molecules. Binding is indicated by a difference in mobility between the gel with ligand present and the gel with no ligand present. The utility of this method was demonstrated using the known interaction between the Escherichia coli ribosomal A-site RNA and different aminoglycoside ligands. The RNA-aminoglycoside interaction observed is dose dependent, and the affinity mirrors what is observed in solution. In addition, we used this method to gauge the affinity to different aminoglycoside molecules of an RNA molecule derived from the thymidylate synthase mRNA construct that contains a CC mismatch. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Separation and quantification of cellulases and hemicellulases by capillary electrophoresis

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Kutter, Jörg Peter; Olsson, Lisbeth

    2003-01-01

    . 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...... silica capillary at pH values close to neutral. The improvement of the separation of these six proteins by the addition of alpha, omega-diaminoalkanes with chain lengths from three to seven carbon units was investigated. Dynamically coating the capillary with 1,3-diaminopropane resulted in separation...... of the six enzymes and the reproducibility of the migration times was between 0.6 and 1.9%. Two cases-quantitative determination of the enzyme concentrations in cultivation samples and investigation of adsorption of the enzymes onto cellulose-demonstrated the advantages and perspectives of CE analysis...

  11. Stacking and discontinuous buffers in capillary zone electrophoresis.

    Science.gov (United States)

    Shihabi, Z K

    2000-08-01

    Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.

  12. Explorative data analysis of two-dimensional electrophoresis gels

    DEFF Research Database (Denmark)

    Schultz, J.; Gottlieb, D.M.; Petersen, Marianne Kjerstine

    2004-01-01

    Methods for classification of two-dimensional (2-DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two-dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening...... of gels is presented. First, an approach is demonstrated in which no prior knowledge of the separated proteins is used. Alignment of the gels followed by a simple transformation of data makes it possible to analyze the gels in an automated explorative manner by principal component analysis, to determine...... if the gels should be further analyzed. A more detailed approach is done by analyzing spot volume lists by principal components analysis and partial least square regression. The use of spot volume data offers a mean to investigate the spot pattern and link the classified protein patterns to distinct spots...

  13. Separation of ions in acidic solution by capillary electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Thornton, Michelle [Iowa State Univ., Ames, IA (United States)

    1997-10-08

    Capillary electrophoresis (CE) is an effective method for separating ionic species according to differences in their electrophoretic mobilities. CE separations of amino acids by direct detection are difficult due to their similar electrophoretic mobilities and low absorbances. However, native amino acids can be separated by CE as cations at a low pH by adding an alkanesulfonic acid to the electrolyte carrier which imparts selectivity to the system. Derivatization is unnecessary when direct UV detection is used at 185 nm. Simultaneous speciation of metal cations such as vanadium (IV) and vanadium (V) can easily be performed without complexation prior to analysis. An indirect UV detection scheme for acidic conditions was also developed using guanidine as the background carrier electrolyte (BCE) for the indirect detection of metal cations. Three chapters have been removed for separate processing. This report contains introductory material, references, and general conclusions. 80 refs.

  14. [Determination of glutamic acid in biological material by capillary electrophoresis].

    Science.gov (United States)

    Narezhnaya, E; Krukier, I; Avrutskaya, V; Degtyareva, A; Igumnova, E A

    2015-01-01

    The conditions for the identification and determination of Glutamic acid by capillary zone electrophoresis without their preliminary derivatization have been optimized. The effect of concentration of buffer electrolyte and pH on determination of Glutamic acid has been investigated. It is shown that the 5 Mm borate buffer concentration and a pH 9.15 are optimal. Quantitative determination of glutamic acid has been carried out using a linear dependence between the concentration of the analyte and the area of the peak. The accuracy and reproducibility of the determination are confirmed by the method "introduced - found". Glutamic acid has been determined in the placenta homogenate. The duration of analysis doesn't exceed 30 minutes. The results showed a decrease in the level of glutamic acid in cases of pregnancy complicated by placental insufficiency compared with the physiological, and this fact allows to consider the level of glutamic acid as a possible marker of complicated pregnancy.

  15. Graphitic carbon nitride embedded hydrogels for enhanced gel electrophoresis.

    Science.gov (United States)

    Zarei, Mohammad; Ahmadzadeh, Hossein; Goharshadi, Elaheh K; Farzaneh, Ali

    2015-08-05

    Here, we show, for the first time, the use of graphitic carbon nitride (g-C3N4) nanosheets to improve the resolution and efficiency of protein separation in gel electrophoresis. By loading 0.04% (m/v) g-C3N4 nanosheets into the polyacrylamide gel at 25 °C, the thermal conductivity increased approximately 80% which resulted in 20% reduction in Joule heating and overall increase of separation efficiency. Also, polymerization of acrylamide occurred in the absence of tetramethylethylenediamine (TEMED) when the polyacrylamide gel contained g-C3N4 nanosheets. Hence, the g-C3N4 act simultaneously as a polymerization catalyst as well as heat sinks to lower Joule heating effect on band broadening. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Liquid crystal-enabled electrophoresis and electro-osmosis

    Science.gov (United States)

    Lavrentovich, Oleg D.

    This work presents a comparative review of electrokinetic effects in isotropic and anisotropic (liquid crystalline) electrolytes. A special emphasis is placed on nonlinear electrokinetics with ow velocities growing as the square of the applied electric field. This phenomenon allows one to drive steady motion of particles and uids with an alternating-current electric field. In isotropic electrolytes, spatial separation of charges that leads to nonlinear electrokinetics is achieved through the properties of the solid component (typically a metal). If the electrolyte is a liquid crystal (LC), its anisotropic properties enable separation of charges in the presence of orientational distortions and under the action of an electric field. LC anisotropy leads to electrically-driven motion of colloidal particles (liquid crystal-enabled electrophoresis, LCEP) and of the LC itself (liquid crystal-enabled electro-osmosis, LCEO). The induced charge is proportional to the applied field, director gradients, anisotropy of conductivity, and anisotropy of permittivity. The electric field acts on the space separated charges to drive the electro-osmotic ows. If the director deformations lack mirror symmetry, the LC enables electrophoresis of free particles and electro-osmotic pumping. The advantage of LCenabled electrokinetics (LCEK) is that its mechanism lifts many restrictions imposed on the properties of the solid counterpart. For example, LCEP can transport particles even if these particles are deprived of any surface charges; the particles can even be a uid immiscible with a LC or a gas bubble. In a similar fashion, LCEO can drive ows even if there are no oating electrodes. Ionic currents in LCs which have been traditionally considered an undesirable feature in displays offer a broad platform for versatile applications in electrokinetics of particles and uids, micropumping and mixing, and lab-on-a-chip analysis...

  18. Measurement of Dissociation Rate of Biomolecular Complexes Using Capillary Electrophoresis

    Science.gov (United States)

    Yang, Peilin; Mao, Yingwei; Lee, Angel W.-M; Kennedy, Robert T.

    2009-01-01

    Fluorescence anisotropy (FA), non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and high-speed capillary electrophoresis (CE) were evaluated for measuring dissociation kinetics of peptide-protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the Src homology 2 (SH2) and SH3 domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of ~53 s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30 cm long. When using Fyn-SH2-SH3 tagged with glutathione S-transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7 s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0 ± 2.7 and 58.8 ± 6.1 s respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that while all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions. PMID:19148904

  19. Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Reza Arjmand

    2015-01-01

    Full Text Available Background: Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM. In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. Materials and Methods: L. major (MRHO/IR/75/ER from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins. Results: Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major. Conclusion: It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.

  20. ['Gold standard', not 'golden standard'

    NARCIS (Netherlands)

    Claassen, J.A.H.R.

    2005-01-01

    In medical literature, both 'gold standard' and 'golden standard' are employed to describe a reference test used for comparison with a novel method. The term 'gold standard' in its current sense in medical research was coined by Rudd in 1979, in reference to the monetary gold standard. In the same