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Sample records for 2d gel electrophoresis

  1. Two-Dimensional Gel Electrophoresis and 2D-DIGE.

    PubMed

    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.

  2. Simultaneous immunoblotting analysis with activity gel electrophoresis and 2-D gel electrophoresis.

    PubMed

    Lee, Der-Yen; Chang, Geen-Dong

    2015-01-01

    Diffusion blotting method can couple immunoblotting analysis with another biochemical technique in a single polyacrylamide gel, however, with lower transfer efficiency as compared to the conventional electroblotting method. Thus, with diffusion blotting, protein blots can be obtained from an SDS polyacrylamide gel for zymography assay, from a native polyacrylamide gel for electrophoretic mobility shift assay (EMSA) or from a 2-D polyacrylamide gel for large-scale screening and identification of a protein marker. Thereafter, a particular signal in zymography, electrophoretic mobility shift assay, and 2-dimensional gel can be confirmed or identified by simultaneous immunoblotting analysis with a corresponding antiserum. These advantages make diffusion blotting desirable when partial loss of transfer efficiency can be tolerated or be compensated by a more sensitive immunodetection reaction using enhanced chemiluminescence detection.

  3. Capabilities using 2-D DIGE in proteomics research : the new gold standard for 2-D gel electrophoresis.

    PubMed

    Rozanas, Christine R; Loyland, Stacey M

    2008-01-01

    The use of two-dimensional gel electrophoresis for differential analysis in proteomics was revolutionized by the introduction of 2-D fluorescence difference gel electrophoresis (2-D DIGE). This fluorescence-based technique allows the use of multiplexed samples and an internal standard that virtually eliminates gel-to-gel variability, resulting in increased confidence that differences found between samples are due to real induced changes, rather than inherent biological variation or experimental variability. 2-D DIGE has quickly become the "gold standard" for gel-based proteomics for studying tissues, as well as cell culture and bodily fluids such as serum or plasma. This chapter will describe the basic 2-D DIGE technique using minimal labeling, image acquisition using high-quality fluorescence scanners, and software capable of analyzing the multiplexed images and normalizing the data using the internal standard.

  4. Protein profiling using two-dimensional difference gel electrophoresis (2-D DIGE).

    PubMed

    Feret, Renata; Lilley, Kathryn S

    2014-02-03

    2-D DIGE relies on pre-electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one 2-D gel. The dye-labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with gel-to-gel variation and spot matching between gels. Image analysis programs are used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable protein abundance level changes to be identified and quantified. This unit describes the 2-D DIGE procedure including sample preparation from various cell types, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples.

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

    PubMed

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

    2017-03-01

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

  6. Optimization of Large Gel 2D Electrophoresis for Proteomic Studies of Skeletal Muscle

    PubMed Central

    Reed, Patrick W.; Densmore, Allison; Bloch, Robert J.

    2013-01-01

    We describe improved methods for large format, 2-dimensional gel electrophoresis (2-DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7M urea + 2 M thiourea, instead of 9M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2D-GE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes. PMID:22589104

  7. 2D Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically Labelled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids such is a common method for investigating the biosynthetic rates of proteins. In this way, the abundance of newly synthesized proteins can be determined by several proteomic techniques including 2D gel electrophoresis (2DE). This chapter describes a protocol for labelling pancreatic islets with (35)S-methionine in the presence of low and high concentrations of glucose, followed by subcellular fractionation enrichment of secretory granule proteins and analysis of the granule protein contents by 2DE. This demonstrated that the biosynthetic rates of most of the granule proteins are co-ordinately regulated in the presence of stimulatory glucose concentrations.

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

    PubMed Central

    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

  9. A Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Protocol for Studies of Neural Precursor Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    This chapter describes the basics of two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of up to distinct proteomes. The example given describes the analysis of undifferentiated and differentiated neural precursor cells labelled with fluorescent Cy3 and Cy5 dyes in comparison to a pooled standard labelled with Cy2. After labelling, the proteomes are mixed together and electrophoresed on the same 2D gels. Scanning the gels at wavelengths specific for each dye allows direct overlay of the two different proteomes and the differences in abundance of specific protein spots can be determined through comparison to the pooled standard.

  10. Optimisation of a 2-D gel electrophoresis protocol for the human-pathogenic fungus Aspergillus fumigatus.

    PubMed

    Kniemeyer, Olaf; Lessing, Franziska; Scheibner, Olaf; Hertweck, Christian; Brakhage, Axel A

    2006-03-01

    Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. One of the important questions concerning A. fumigatus is the identification of pathogenicity determinants. To obtain a comprehensive overview about the proteins produced at different physiological conditions that are related to the infectious process a proteomic approach has been applied. Here, 2-D gel electrophoresis for filamentous fungi was optimised concerning removal of interfering compounds, protein extraction and separation methods. A trichloroacetic acid-based precipitation method of proteins with their subsequent solubilisation by the use of a combination of CHAPS with a second sulfobetaine detergent gave the best results. The optimised protocol was evaluated by the analysis of the proteomes of A. fumigatus grown on two different carbon sources, i.e., glucose and ethanol. Carbon catabolite repression has not been studied in detail at the protein level in A. fumigatus yet. In addition, growth on ethanol leads to activation of the glyoxylate cycle which was shown to be essential for pathogenesis in bacteria and fungi. In A. fumigatus, differential patterns of enzymes of the gluconeogenesis, glyoxylate cycle and ethanol degradation pathway during growth on glucose and ethanol were observed.

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

    ERIC Educational Resources Information Center

    Kim, Thomas D.; Craig, Paul A.

    2010-01-01

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

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

    ERIC Educational Resources Information Center

    Kim, Thomas D.; Craig, Paul A.

    2010-01-01

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

  13. Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle.

    PubMed

    Reed, Patrick W; Densmore, Allison; Bloch, Robert J

    2012-04-01

    We describe improved methods for large format, two-dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes.

  14. Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.

    PubMed

    Lohnes, Karen; Quebbemann, Neil R; Liu, Kate; Kobzeff, Fred; Loo, Joseph A; Ogorzalek Loo, Rachel R

    2016-07-15

    The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein 'A' on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated 'top-down' MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of 'top-down' and 'bottom-up' proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Proteomic investigation of human burn wounds by 2D-difference gel electrophoresis and mass spectrometry

    PubMed Central

    Pollins, Alonda C.; Friedman, David B.; Nanney, Lillian B.

    2009-01-01

    Background In humans, thermal cutaneous injury represents a serious traumatic event that induces a host of dynamic alterations. Unfortunately the molecular mechanisms that underlie these serious perturbations remain poorly understood. We applied a global analysis method to identify dynamically changing proteins within the burn environment, which could eventually become biomarkers or targets for treatment. Materials and Methods Protein extracts of normal/unwounded skin and burn wounds were assayed by 2D difference gel electrophoresis (DIGE), a proteomic technology by which abundance levels of intact proteins (including isoforms) were simultaneously quantified from multiple samples with statistical confidence. Through unsupervised multivariate principal component analysis, protein expression patterns from individual samples were appropriately clustered into their correct temporal healing periods grouped into postburn periods of 1–3 days, 4–6 days or 7–10 days after injury. Forty-six proteins were subsequently selected for identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Results Proteins identified with differential temporal patterns of expression included predictable cytoskeletal proteins such as vimentin, and keratins 1, 5, 6, 16, and 17. Other candidate proteins with potential involvement in healing included HSP90, members of the serpin family (Serpin B1, SCCA1 & 2), haptoglobin, gelsolin, eIF4A1, IQGAP1, and TCTP. Conclusions We have utilized the combined technique, DIGE/MS, to capture new insights into cutaneous responses to burn trauma and subsequent processes of early wound healing in humans. This pilot study provides a proteomic snapshot of temporal events that can be used to weave together the interconnected processes that define the response to serious cutaneous injury. PMID:17604053

  16. Identification of Methanococcus Jannaschii Proteins in 2-D Gel Electrophoresis Patterns by Mass Spectrometry

    DOE R&D Accomplishments Database

    Liang, X.

    1998-06-10

    The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.

  17. Analysis of Telomere-Homologous DNA with Different Conformations Using 2D Agarose Electrophoresis and In-Gel Hybridization.

    PubMed

    Zhang, Zepeng; Hu, Qian; Zhao, Yong

    2017-01-01

    In mammalian cells, in addition to double-stranded telomeric DNA at chromosome ends, extra telomere-homologous DNA is present that adopts different conformations, including single-stranded G- or C-rich DNA, extrachromosomal circular DNA (T-circle), and telomeric complex (T-complex) with an unidentified structure. The formation of such telomere-homologous DNA is closely related to telomeric DNA metabolism and chromosome end protection by telomeres. Conventional agarose gel electrophoresis is unable to separate DNA based on conformation. Here, we introduce the method of two-dimensional (2D) agarose electrophoresis in combination with in-gel native/denatured hybridization to determine different conformations formed by telomere-homologous DNA.

  18. Development of an open source laboratory information management system for 2-D gel electrophoresis-based proteomics workflow

    PubMed Central

    Morisawa, Hiraku; Hirota, Mikako; Toda, Tosifusa

    2006-01-01

    Background In the post-genome era, most research scientists working in the field of proteomics are confronted with difficulties in management of large volumes of data, which they are required to keep in formats suitable for subsequent data mining. Therefore, a well-developed open source laboratory information management system (LIMS) should be available for their proteomics research studies. Results We developed an open source LIMS appropriately customized for 2-D gel electrophoresis-based proteomics workflow. The main features of its design are compactness, flexibility and connectivity to public databases. It supports the handling of data imported from mass spectrometry software and 2-D gel image analysis software. The LIMS is equipped with the same input interface for 2-D gel information as a clickable map on public 2DPAGE databases. The LIMS allows researchers to follow their own experimental procedures by reviewing the illustrations of 2-D gel maps and well layouts on the digestion plates and MS sample plates. Conclusion Our new open source LIMS is now available as a basic model for proteome informatics, and is accessible for further improvement. We hope that many research scientists working in the field of proteomics will evaluate our LIMS and suggest ways in which it can be improved. PMID:17018156

  19. Quality control and stability studies with the monoclonal antibody, trastuzumab: application of 1D- vs. 2D-gel electrophoresis.

    PubMed

    Nebija, Dashnor; Noe, Christian R; Urban, Ernst; Lachmann, Bodo

    2014-04-15

    Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.

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

    PubMed Central

    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

  1. Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis.

    PubMed

    Mohannath, Gireesha; Pikaard, Craig S

    2016-01-01

    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-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.

  2. Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE).

    PubMed

    Gao, Liyan; Wang, Aili; Li, Xiaohui; Dong, Kun; Wang, Ke; Appels, Rudi; Ma, Wujun; Yan, Yueming

    2009-12-01

    Comparative proteomics analysis offers a new approach to identify differential proteins among different wheat genotypes and developmental stages. In this study, the non-prolamin expression profiles during grain development of two common or bread wheat cultivars (Triticum aestivum L.), Jing 411 and Sunstate, with different quality properties were analyzed using two-dimensional difference gel electrophoresis (2-D DIGE). Five grain developmental stages during the post-anthesis period were sampled corresponding to the cumulative averages of daily temperatures ( degrees C: 156 degrees C, 250 degrees C, 354 degrees C, 447 degrees C and 749.5 degrees C). More than 400 differential protein spots detected at one or more of the developmental stages of the two cultivars were monitored, among which 230 proteins were identified by MS. Of the identified proteins, more than 85% were enzymes possessing different physiological functions. A total of 36 differential proteins were characterized between the two varieties, which are likely to be related to wheat quality attributes. About one quarter of the proteins identified expressed in multiple spots with different pIs and molecular masses, implying certain post-translational modifications (PTMs) of proteins such as phosphorylations and glycosylations. The results provide new insights into biochemical mechanisms for grain development and quality.

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

    PubMed

    Singh, Nisha; Jain, Neha; Kumar, Ram; Jain, Ajay; Singh, Nagendra K; Rai, Vandna

    2015-01-01

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

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

    PubMed Central

    Singh, Nisha; Jain, Neha; Kumar, Ram; Jain, Ajay; Singh, Nagendra K.; Rai, Vandna

    2015-01-01

    Pigeonpea is an important legume crop with high protein content. However, it is often subjected to various abiotic and biotic stresses. Proteomics is a state-of-the-art technique used to analyze the protein profiling of a tissue for deciphering the molecular entities that could be manipulated for developing crops resistant to these stresses. In this context, developing a comprehensive proteome profile from different vegetative and reproductive tissues has become mandatory. Although several protein extraction protocols from different tissues of diverse plant species have been reported, there is no report for pigeonpea. Here, we report tissue-specific protein extraction protocols representing vegetative (young leaves), and reproductive (flowers and seeds) organs and their subsequent analysis on 2-dimensional gel electrophoresis. The study explicitly demonstrated that the efficacy of a particular protein extraction protocol is dependent on the different tissues, such as leaves, flowers and seeds that differ in their structure and metabolic constituents. For instance, phenol-based protocol showed an efficacy toward higher protein yield, better spot resolution and a minimal streaking on 2-DE gel for both leaves and flowers. Protein extraction from seeds was best achieved by employing phosphate-TCA-acetone protocol. PMID:26300903

  5. Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IEF of Proteins in an IEF-Dedicated Electrophoresis Unit.

    PubMed

    Stochaj, Wayne R; Berkelman, Tom; Laird, Nancy

    2006-10-01

    INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels, providing the first dimension of the 2D separation. In this protocol, the IPG gels are focused using self-contained instruments for IEF. These high-voltage systems allow fewer manipulations of the IPG gels, resulting in less error, strip mix-up, contamination, air contact, or urea crystallization. Because rehydration and IEF can be performed consecutively within a single unit, these two steps can be performed unattended overnight. Finally, faster separations and sharper focusing are possible due to the higher voltage available in these instruments.

  6. Metal imaging in non-denaturating 2D electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for the detection of metalloproteins.

    PubMed

    Becker, J Susanne; Lobinski, Ryszard; Becker, J Sabine

    2009-01-01

    Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed as a powerful analytical technique for metal imaging of 2D gels for the detection of metalloproteins in rat kidney after electrophoretic separation. Protein complexes, extracted with water, were separated in their native state in the first and second dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, manganese and lead, were monitored by LA-ICP-MS after gel ablation by a focused laser beam in a way that the total surface of a selected fragment of the gel was totally ablated. The metal distribution of this part of the gel was then constructed by plotting the metal (isotope) signal intensity as a function of the x,y (isoelectric point, molecular mass) coordinates of the gel. The proteins at locations rich in metals were cut out, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

  7. Pulse Field Gel Electrophoresis

    PubMed Central

    Sharma-Kuinkel, Batu K.; Rude, Thomas H.; Fowler, Vance G.

    2015-01-01

    Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments. PMID:25682374

  8. Pulse Field Gel Electrophoresis.

    PubMed

    Sharma-Kuinkel, Batu K; Rude, Thomas H; Fowler, Vance G

    2016-01-01

    Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments.

  9. Agarose gel electrophoresis.

    PubMed

    Smith, D R

    1993-01-01

    After digestion of DNA with a restriction enzyme (Chapter 50), it is usually necessary, for both preparative and analytical purposes, to separate and visualize the products. In most cases, where the products are between 200 and 20,000 bp long, this is achieved by agarose gel electrophoresis. Agarose is a linear polymer that is extracted from seaweed and sold as a white powder. The powder is melted in buffer and allowed to cool, whereby the agarose forms a gel by hydrogen bonding. The hardened matrix contains pores, the size of which depends on the concentration of agarose. The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v), and agarose gels are normally in the range of 0.3 to 3%. Many different apparatus arrangements have been devised to run agarose gels; for example, they can be run horizontally or vertically, and the current can be conducted by wicks or the buffer solution. However, today, the "submarine" gel system is almost universally used. In this method, the agarose gel is formed on a supporting plate, and then the plate is submerged into a tank containing a suitable electrophoresis buffer. Wells are preformed in the agarose gel with the aid of a "comb" that is inserted into the cooling agarose before the agarose has gelled. Into these wells are loaded the sample to be analyzed, which has been mixed with a dense solution (a loading buffer) to ensure that the sample sinks into the wells.

  10. Comparison of the urinary protein patterns of athletes by 2D-gel electrophoresis and mass spectrometry-a pilot study.

    PubMed

    Kohler, Maxie; Franz, Stefan; Regeniter, Axel; Ikonen, Anna; Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2009-08-01

    Urinary proteins and exercise-induced proteinuria have been the subject of much research. Proteinuria has been studied in depth after different running and cycling intensities and durations and the different mechanisms of glomerular filtration and tubular dysfunction have been elucidated. The present study was carried out to compare urinary protein profiles of athletes in different sport categories (endurance sport, team sport, strength sport). Doping-control urine samples obtained from in-competition testing and specimens derived from a control group were analysed by means of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and significantly deviating protein spots were enzymatically hydrolysed and identified by nanoflow liquid chromatography-orbitrap mass spectrometry. Endurance sport samples demonstrated a significant increase of mainly medium-sized urinary proteins such as transferrin, zinc alpha-2-glycoprotein and prostaglandin H2 D-isomerase (30-80 kDa) in 2D-PAGE experiments. Proteinuria was evident in all samples after protein concentration measurements (protein/creatinine > 15 mg/mmol). Alterations were also observed in strength sport samples, which showed an increase of low molecular weight proteins or protein fragments (<30 kDa, e.g., transthyretin, CD 59 antigen or an N-terminal transferrin fragment). In contrast, the concentration measurements did not imply proteinuria but total protein excretion was in a normal range. The study provides a first overview on 2D maps of the urinary proteome after different types of exercise. Future studies may lead to the establishment of urinary protein maps that are typical for a certain type of sport or even an individual athlete. These maps may complement the blood passport of athletes in doping control.

  11. Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes

    PubMed Central

    2013-01-01

    Background Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well. Results To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. Conclusions The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co

  12. 2-D difference gel electrophoresis approach to assess protein expression profiles in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vents.

    PubMed

    Company, Rui; Antúnez, Oreto; Bebianno, Maria João; Cajaraville, Miren P; Torreblanca, Amparo

    2011-11-18

    Hydrothermal vent mussels Bathymodiolus azoricus are naturally exposed to toxic chemical species originated directly from vent chimneys. The amount of toxic elements varies significantly among vent sites along the Mid-Atlantic Ridge and B. azoricus must be able to adapt to changes in hydrothermal fluid composition, temperature and pressure. The aim of this work was to study changes in the proteome in the "gill-bacteria complex" of mussels B. azoricus from three hydrothermal vent sites with distinct environmental characteristics using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Results showed that 31 proteins had different expression profiles among vent sites and both cluster and principal component analysis confirm a clear separation of mussels between sites. This suggests the existence of specific parameters grouping individuals from the same hydrothermal site. Protein spots of the more abundant differentially expressed proteins were excised, digested with trypsin and identified by mass spectrometry. All identified proteins (actin, ubiquinone, S-adenosylhomocysteine hydrolase, cysteine peptidases, chaperonin and catalase) have been related previously with oxidative stress conditions and are known to be affected by ROS inducing stressors, including metals. Results point out to specific adaptations at the proteome level of B. azoricus depending on the level of toxicants present in their environment. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2015-04-01

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

  14. Polyacrylamide gel electrophoresis.

    PubMed

    Chrambach, A; Rodbard, D

    1971-04-30

    Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain reproducible relative mobility (Rf) values as physical-chemical constants. Application and extension of Ogston's (random fiber) model for a gel allows for calculation of molecular volume, surface area, or radius, free mobility, and valence from RJ measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking) with known operating characteristics for any pH at 0 degrees and 25 degrees C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative scale PAGE is operational for milligram loads and feasible for gram quantities.

  15. Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics.

    PubMed

    Kondo, Tadashi; Hirohashi, Setsuo

    2006-01-01

    Proteome data combined with histopathological information provides important, novel clues for understanding cancer biology and reveals candidates for tumor markers and therapeutic targets. We have established an application of a highly sensitive fluorescent dye (CyDye DIGE Fluor saturation dye), developed for two-dimensional difference gel electrophoresis (2D-DIGE), to the labeling of proteins extracted from laser microdissected tissues. The use of the dye dramatically decreases the protein amount and, in turn, the number of cells required for 2D-DIGE; the cells obtained from a 1 mm2 area of an 8-12 microm thick tissue section generate up to 5,000 protein spots in a large-format 2D gel. This protocol allows the execution of large-scale proteomics in a more efficient, accurate and reproducible way. The protocol can be used to examine a single sample in 5 d or to examine hundreds of samples in large-scale proteomics.

  16. Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry.

    PubMed

    Hanna, S L; Sherman, N E; Kinter, M T; Goldberg, J B

    2000-10-01

    Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (microLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by microLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.

  17. Copolymers For Capillary Gel Electrophoresis

    DOEpatents

    Liu, Changsheng; Li, Qingbo

    2005-08-09

    This invention relates to an electrophoresis separation medium having a gel matrix of at least one random, linear copolymer comprising a primary comonomer and at least one secondary comonomer, wherein the comonomers are randomly distributed along the copolymer chain. The primary comonomer is an acrylamide or an acrylamide derivative that provides the primary physical, chemical, and sieving properties of the gel matrix. The at least one secondary comonomer imparts an inherent physical, chemical, or sieving property to the copolymer chain. The primary and secondary comonomers are present in a ratio sufficient to induce desired properties that optimize electrophoresis performance. The invention also relates to a method of separating a mixture of biological molecules using this gel matrix, a method of preparing the novel electrophoresis separation medium, and a capillary tube filled with the electrophoresis separation medium.

  18. Comparative Two-Dimensional Fluorescence Gel Electrophoresis.

    PubMed

    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.

  19. Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

    PubMed

    Xu, Ming; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra

    2014-10-01

    Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism.

  20. Normalization and expression changes in predefined sets of proteins using 2D gel electrophoresis: A proteomic study of L-DOPA induced dyskinesia in an animal model of Parkinson's disease using DIGE

    PubMed Central

    Kultima, Kim; Scholz, Birger; Alm, Henrik; Sköld, Karl; Svensson, Marcus; Crossman, Alan R; Bezard, Erwan; Andrén, Per E; Lönnstedt, Ingrid

    2006-01-01

    Background Two-Dimensional Difference In Gel Electrophoresis (2D-DIGE) is a powerful tool for measuring differences in protein expression between samples or conditions. However, to remove systematic variability within and between gels the data has to be normalized. In this study we examined the ability of four existing and four novel normalization methods to remove systematic bias in data produced with 2D-DIGE. We also propose a modification of an existing method where the statistical framework determines whether a set of proteins shows an association with the predefined phenotypes of interest. This method was applied to our data generated from a monkey model (Macaca fascicularis) of Parkinson's disease. Results Using 2D-DIGE we analysed the protein content of the striatum from 6 control and 21 MPTP-treated monkeys, with or without de novo or long-term L-DOPA administration. There was an intensity and spatial bias in the data of all the gels examined in this study. Only two of the eight normalization methods evaluated ('2D loess+scale' and 'SC-2D+quantile') successfully removed both the intensity and spatial bias. In 'SC-2D+quantile' we extended the commonly used loess normalization method against dye bias in two-channel microarray systems to suit systems with three or more channels. Further, by using the proposed method, Differential Expression in Predefined Proteins Sets (DEPPS), several sets of proteins associated with the priming effects of L-DOPA in the striatum in parkinsonian animals were identified. Three of these sets are proteins involved in energy metabolism and one set involved proteins which are part of the microtubule cytoskeleton. Conclusion Comparison of the different methods leads to a series of methodological recommendations for the normalization and the analysis of data, depending on the experimental design. Due to the nature of 2D-DIGE data we recommend that the p-values obtained in significance tests should be used as rankings only. Individual

  1. Quantitation of protein in samples prepared for 2-D electrophoresis.

    PubMed

    Berkelman, Tom

    2008-01-01

    The concentration of protein in a sample prepared for two dimensional (2-D) electrophoretic analysis is usually determined by protein assay. Reasons for this include the following. (1) Protein quantitation ensures that the amount of protein to be separated is appropriate for the gel size and visualization method. (2) Protein quantitation facilitates comparison among similar samples, as image-based analysis is simplified when equivalent quantities of proteins have been loaded on the gels to be compared. (3) Quantitation is necessary in cases where the protein sample is labeled with dye before separation (1,2). The labeling chemistry is affected by the dye to protein ratio so it is essential to know the protein concentration before setting up the labeling reaction.A primary consideration with quantitating protein in samples prepared for 2-D electrophoresis is interference by nonprotein substances that may be present in the sample. These samples generally contain chaotropic solubilizing agents, detergents, reductants, buffers or carrier ampholytes, all of which potentially interfere with protein quantitation. The most commonly used protein assays in proteomics research are colorimetric assays in which the presence of protein causes a color change that can be measured spectrophotometrically (3). All protein assays utilize standards, a dilution series of a known concentration of a known protein, to create a standard curve. Two methods will be considered that circumvent some of the problems associated with interfering substances and are well suited for samples prepared for 2-D electrophoresis. The first method (4.1.1) relies on a color change that occurs upon binding of a dye to protein and the second (4.1.2) relies on binding and reduction of cupric ion (Cu2+) ion to cuprous ion (Cu+) by proteins.

  2. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    EPA Science Inventory

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  3. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    EPA Science Inventory

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  4. Separation of basic proteins from Leishmania using a combination of Free flow electrophoresis (FFE) and 2D electrophoresis (2-DE) under basic conditions.

    PubMed

    Brotherton, Marie-Christine; Racine, Gina; Ouellette, Marc

    2015-01-01

    Basic proteins, an important class of proteins in intracellular organisms such as Leishmania, are usually underrepresented on 2D gels. This chapter describes a method combining basic proteins fractionation using Free flow electrophoresis in isoelectric focusing mode (IEF-FFE) followed by protein separation using two-dimensional gel electrophoresis (2-DE) in basic conditions. The combination of these two techniques represents a great improvement for the visualization of Leishmania proteins with basic pI using 2D gels.

  5. Analysis of branched DNA replication and recombination intermediates from prokaryotic cells by two-dimensional (2D) native-native agarose gel electrophoresis.

    PubMed

    Robinson, Nicholas P

    2013-01-01

    Branched DNA molecules are generated by the essential processes of replication and recombination. Owing to their distinctive extended shapes, these intermediates migrate differently from linear double-stranded DNA under certain electrophoretic conditions. However, these branched species exist in the cell at much low abundance than the bulk linear DNA. Consequently, branched molecules cannot be visualized by conventional electrophoresis and ethidium bromide staining. Two-dimensional native-native agarose electrophoresis has therefore been developed as a method to facilitate the separation and visualization of branched replication and recombination intermediates. A wide variety of studies have employed this technique to examine branched molecules in eukaryotic, archaeal, and bacterial cells, providing valuable insights into how DNA is duplicated and repaired in all three domains of life.

  6. Conducting Polymer Electrodes for Gel Electrophoresis

    PubMed Central

    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. PMID:24586761

  7. Conducting polymer electrodes for gel electrophoresis.

    PubMed

    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.

  8. Proteome analysis of responses to ascochlorin in a human osteosarcoma cell line by 2-D gel electrophoresis and MALDI-TOF MS.

    PubMed

    Kang, Jeong Han; Park, Kwan-Kyu; Lee, In-Seon; Magae, Junji; Ando, Kunio; Kim, Cheorl-Ho; Chang, Young-Chae

    2006-10-01

    Ascochlorin is a prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. Ascochlorin reduces serum cholesterol and triglyceride levels, suppresses hypertension and tumor development, and ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ascochlorin regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the proteome of the human osteosarcoma cells U2OS in response to ascochlorin. In addition, we established the first two-dimensional map of the U2OS proteome. The U2OS cell proteomes with and without treatment with ascochlorin were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and bioinformatics. The largest differences in expression were observed for the epidermal growth factor receptor (4-fold decrease), ribulose-5-phosphate-epimerase (13-fold decrease), ATP-dependent RNA helicase (8-fold decrease), and kelch-like ECH-associated protein 1 (6-fold decrease). The abundance of heterogeneous nuclear ribonucleoprotein L and minichromosome maintenance protein 7 increased 12- and 8.2-fold, respectively. In addition, Erk 2 was increased 3-fold in U2OS cells treated with ascochlorin. The expression of some selected proteins was confirmed by western blotting, zymography and RT-PCR analysis.

  9. Fluorescence detection for gel and capillary electrophoresis

    SciTech Connect

    Hogan, B.

    1992-07-21

    First, an indirect fluorescence detection system for the separation of proteins via gel electrophoresis. Quantities as low as 50 nanograms of bovine serum albumin and soybean trypsin inhibitor are separated and detected visually without the need for staining of the analytes. This is very similar to levels of protein commonly separated with gel electrophoresis.

  10. A Simple Vertical Slab Gel Electrophoresis Apparatus.

    ERIC Educational Resources Information Center

    Carter, J. B.; And Others

    1983-01-01

    Describes an inexpensive, easily constructed, and safe vertical slab gel kit used routinely for sodium dodecyl sulphate-polyacrylamide gel electrophoresis research and student experiments. Five kits are run from a single transformer. Because toxic solutions are used, students are given plastic gloves and closely supervised during laboratory…

  11. SDS-Polyacrylamide Gel Electrophoresis of Proteins.

    PubMed

    Sambrook, Joseph; Russell, David W

    2006-09-01

    INTRODUCTIONThis protocol describes the separation of proteins by SDS-polyacrylamide gel electrophoresis. SDS is used with a reducing agent and heat to dissociate the proteins. SDS-polypeptide complexes form and migrate through the gels according to the size of the polypeptide. By using markers of known molecular weight, the molecular weight of the polypeptide chain(s) can be estimated.

  12. A Simple Vertical Slab Gel Electrophoresis Apparatus.

    ERIC Educational Resources Information Center

    Carter, J. B.; And Others

    1983-01-01

    Describes an inexpensive, easily constructed, and safe vertical slab gel kit used routinely for sodium dodecyl sulphate-polyacrylamide gel electrophoresis research and student experiments. Five kits are run from a single transformer. Because toxic solutions are used, students are given plastic gloves and closely supervised during laboratory…

  13. Supramolecular gel electrophoresis of large DNA fragments.

    PubMed

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

    2017-07-06

    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. C3 -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.

  14. Electrokinetics of nanoparticle gel-electrophoresis.

    PubMed

    Hill, Reghan J

    2016-09-28

    Gel-electrophoresis has been demonstrated in recent decades to successfully sort a great variety of nanoparticles according to their size, charge, surface chemistry, and corona architecture. However, quantitative theoretical interpetations have been limited by the number and complexity of factors that influence particle migration. Theoretical models have been fragmented and incomplete with respect to their counterparts for free-solution electrophoresis. This paper unifies electrokinetic models that address complex nanoparticle corona architectures, corona and gel charge regulation (e.g., by the local pH), multi-component electrolytes, and non-linear electrostatics and relaxation effects. By comprehensively addressing the electrokinetic aspects of the more general gel-electrophoresis problem, in which short-ranged steric interactions are significant, a stage is set to better focus on the physicochemical and steric factors. In this manner, it is envisioned that noparticle gel-electrophoresis may eventually be advanced from a nanoparticle-characterization tool to one that explicitly probes the short-ranged interactions of nanoparticles with soft networks, such as synthetic gels and biological tissues. In this paper, calculations are undertaken that identify a generalized Hückel limit for nanoparticles in low-conductivity gels, and a new Smoluchowski limit for polyelectrolyte-coated particles in high-conductivity gels that is independent of the gel permeability. Also of fundamental interest is a finite, albeit small, electrophoretic mobility for uncharged particles in charged gels. Electrophoretic mobilities and drag coefficients (with electroviscous effects) for nanoparticles bearing non-uniform coronas show that relaxation effects are typically weak for the small nanoparticles (radius ≈3-10 nm) to which gel-electrophoresis has customarily been applied, but are profound for the larger nanoparticles (radius ≳ 40 nm in low conductivity gels) to which passivated gel-electrophoresis

  15. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    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.

  16. Nondenaturing agarose gel electrophoresis of RNA.

    PubMed

    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, denaturing acrylamide gels are most appropriate. In contrast, agarose gels are generally used to analyze 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.

  17. Nonlinear gel electrophoresis: an analogy with ideal fluid flow.

    PubMed

    Dennison, C; Phillips, A M; Nevin, J M

    1983-12-01

    The behavior of electrolytes undergoing electrophoresis in various shaped gels was investigated using bromphenol blue as a model electrolyte. The results suggest that during gel electrophoresis, small electrolytes behave in a manner analogous to the flow of ideal, irrotational fluids.

  18. Comparative proteomics and difference gel electrophoresis.

    PubMed

    Minden, Jonathan

    2007-12-01

    The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference gel electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE.

  19. pI-Control in Comparative Fluorescence Gel Electrophoresis (CoFGE) using amphoteric azo dyes

    PubMed Central

    Hanneken, Marina; Šlais, Karel; König, Simone

    2015-01-01

    Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates. PMID:26217748

  20. pI-Control in Comparative Fluorescence Gel Electrophoresis (CoFGE) using amphoteric azo dyes.

    PubMed

    Hanneken, Marina; Šlais, Karel; König, Simone

    2015-06-01

    Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates.

  1. Gel Electrophoresis of Gold-DNA Nanoconjugates

    DOE PAGES

    Pellegrino, T.; Sperling, R. A.; Alivisatos, A. P.; ...

    2007-01-01

    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 effectivemore » 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.« less

  2. Hybrid slab-microchannel gel electrophoresis system

    DOEpatents

    Balch, Joseph W.; Carrano, Anthony V.; Davidson, James C.; Koo, Jackson C.

    1998-01-01

    A hybrid slab-microchannel gel electrophoresis system. The hybrid system permits the fabrication of isolated microchannels for biomolecule separations without imposing the constraint of a totally sealed system. The hybrid system is reusable and ultimately much simpler and less costly to manufacture than a closed channel plate system. The hybrid system incorporates a microslab portion of the separation medium above the microchannels, thus at least substantially reducing the possibility of non-uniform field distribution and breakdown due to uncontrollable leakage. A microslab of the sieving matrix is built into the system by using plastic spacer materials and is used to uniformly couple the top plate with the bottom microchannel plate.

  3. Multifluorescence 2D gel imaging and image analysis.

    PubMed

    Vormbrock, Ingo; Hartwig, Sonja; Lehr, Stefan

    2012-01-01

    Although image acquisition and analysis are crucial steps within the multifluorescence two-dimensional gel electrophoresis workflow, some basics are frequently not carried out with the necessary diligence. This chapter should help to prevent easily avoidable failures during imaging and image preparation for comparative protein analysis.

  4. Identification and In Silico Analysis of Major Redox Modulated Proteins from Brassica juncea Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).

    PubMed

    Chaurasia, Satya Prakash; Deswal, Renu

    2017-02-01

    The thiol-disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H2O2) induced changes in the gene expression have been reported, but efforts to detect H2O2 modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol-disulphide exchange in Brassica juncea seedlings following H2O2 (10 mM) treatment for 30 min. Eleven redox responsive polypeptides were identified which included cruciferin, NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)] interacting protein phosphatase, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit, and myrosinase. Redox modulation of RuBisCO large subunit was further confirmed by western blotting. However, the small subunit of RuBisCO was not affected by these redox changes. All redox modulated targets except NLI interacting protein (although it contains two cysteines) showed oxidation sensitive cysteines by in silico analysis. Interestingly, interactome of cruciferin and myrosinase indicated that they may have additional function(s) beside their well-known roles in the seedling development and abiotic stress respectively. Cruciferin showed interactions with stress associated proteins like defensing-like protein 192 and 2-cys peroxiredoxin. Similarly, myrosinase showed interactions with nitrilase and cytochrome p450 which are involved in nitrogen metabolism and/or hormone biosynthesis. This simple procedure can be used to detect major stress mediated redox changes in other plants.

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

    ERIC Educational Resources Information Center

    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)

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

    ERIC Educational Resources Information Center

    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)

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

    PubMed

    Tsai, Henghang; Leung, Hon-Chiu Eastwood

    2012-01-01

    Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and nonspecific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein recovery efficiency. Here, we describe the enhancement of protein separation efficiency up to twofold in conventional one-dimensional PAG electrophoresis (1D PAGE), two-dimensional (2D) PAGE, and native PAGE by implementing pulses of inverted electric field during gel electrophoresis.

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

    PubMed

    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.

  9. 2D electrophoresis-based expression proteomics: a microbiologist's perspective.

    PubMed

    Sá-Correia, Isabel; Teixeira, Miguel C

    2010-12-01

    Quantitative proteomics based on 2D electrophoresis (2-DE) coupled with peptide mass fingerprinting is still one of the most widely used quantitative proteomics approaches in microbiology research. Our view on the exploitation of this global expression analysis technique and its contribution and potential to push forward the field of molecular microbial physiology towards a molecular systems microbiology perspective is discussed in this article. The advances registered in 2-DE-based quantitative proteomic analysis leading to increased protein resolution, sensitivity and accuracy, and the promising use of 2-DE to gain insights into post-translational modifications at a proteome-wide level (considering all the proteins/protein forms expressed by the genome) are focused on. Given the progress made in this field, it is foreseen that the 2-DE-based approach to quantitative proteomics will continue to be a fundamental tool for microbiologists working at a genome-wide scale. Guidelines are also provided for the exploitation of expression proteomics data, based on useful computational tools, and for the integration of these data with other genome-wide expression information. The advantages and limitations of a complete 2-DE-based expression proteomics analysis, envisaging the quantification of the global changes occurring in the proteome of a given cell depending on environmental or genetic manipulations, are discussed from the microbiologist's perspective. Particular focus is given to the emerging field of toxicoproteomics, a new systems toxicity approach that offers a powerful tool to directly monitor the earliest stages of the toxicological response by identifying critical proteins and pathways that are affected by, and respond to, a chemical stress. The experimental design and the bioinformatics analysis of data used in our laboratory to gain mechanistic insights through expression proteomics into the responses of the eukaryotic model Saccharomyces cerevisiae or of

  10. Plasmid DNA topology assayed by two-dimensional agarose gel electrophoresis.

    PubMed

    Schvartzman, Jorge B; Martínez-Robles, María-Luisa; Hernández, Pablo; Krimer, Dora B

    2013-01-01

    Two-dimensional (2D) agarose gel electrophoresis is nowadays one of the best methods available to analyze DNA molecules with different masses and shapes. The possibility to use nicking enzymes and intercalating agents to change the twist of DNA during only one or in both runs, improves the capacity of 2D gels to discern molecules that apparently may look alike. Here we present protocols where 2D gels are used to understand the structure of DNA molecules and its dynamics in living cells. This knowledge is essential to comprehend how DNA topology affects and is affected by all the essential functions that DNA is involved in: replication, transcription, repair and recombination.

  11. Hybrid slab-microchannel gel electrophoresis system

    DOEpatents

    Balch, J.W.; Carrano, A.V.; Davidson, J.C.; Koo, J.C.

    1998-05-05

    A hybrid slab-microchannel gel electrophoresis system is described. The hybrid system permits the fabrication of isolated microchannels for biomolecule separations without imposing the constraint of a totally sealed system. The hybrid system is reusable and ultimately much simpler and less costly to manufacture than a closed channel plate system. The hybrid system incorporates a microslab portion of the separation medium above the microchannels, thus at least substantially reducing the possibility of non-uniform field distribution and breakdown due to uncontrollable leakage. A microslab of the sieving matrix is built into the system by using plastic spacer materials and is used to uniformly couple the top plate with the bottom microchannel plate. 4 figs.

  12. The gel electrophoresis markup language (GelML) from the Proteomics Standards Initiative.

    PubMed

    Gibson, Frank; Hoogland, Christine; Martinez-Bartolomé, Salvador; Medina-Aunon, J Alberto; Albar, Juan Pablo; Babnigg, Gyorgy; Wipat, Anil; Hermjakob, Henning; Almeida, Jonas S; Stanislaus, Romesh; Paton, Norman W; Jones, Andrew R

    2010-09-01

    The Human Proteome Organisation's Proteomics Standards Initiative has developed the GelML (gel electrophoresis markup language) data exchange format for representing gel electrophoresis experiments performed in proteomics investigations. The format closely follows the reporting guidelines for gel electrophoresis, which are part of the Minimum Information About a Proteomics Experiment (MIAPE) set of modules. GelML supports the capture of metadata (such as experimental protocols) and data (such as gel images) resulting from gel electrophoresis so that laboratories can be compliant with the MIAPE Gel Electrophoresis guidelines, while allowing such data sets to be exchanged or downloaded from public repositories. The format is sufficiently flexible to capture data from a broad range of experimental processes, and complements other PSI formats for MS data and the results of protein and peptide identifications to capture entire gel-based proteome workflows. GelML has resulted from the open standardisation process of PSI consisting of both public consultation and anonymous review of the specifications.

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

    PubMed

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

    2015-01-01

    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. 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. 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. 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.

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

    PubMed Central

    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

  15. Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials

    ERIC Educational Resources Information Center

    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…

  16. Inexpensive and Safe DNA Gel Electrophoresis Using Household Materials

    ERIC Educational Resources Information Center

    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. An update on conformation sensitive gel electrophoresis.

    PubMed

    Ganguly, Arupa

    2002-04-01

    Conformation-sensitive gel electrophoresis (CSGE) was developed as a method of heteroduplex analysis to screen large multi-exon genes for sequence variation. The novelty of the method was in the use of a non-proprietary acrylamide gel matrix that used 1,4-bis (acrolyl) piperazine (BAP) as a cross linker with ethylene glycol and formamide as mildly denaturing solvents. The denaturing environment enhances the conformation polymorphism present in DNA heteroduplexes containing variations as small as single nucleotide polymorphisms (SNPs). CSGE has also been adapted for use on a fluorescent platform (F-CSGE) that resulted in higher throughput and sensitivity. Variation in sensitivity of CSGE has been studied extensively. The results demonstrate that the nature of the mismatched base in a defined sequence context has the most profound effect on the conformation of the heteroduplex. Additionally, the size of the PCR product, as well as the location of the mismatch within the PCR product, are two important parameters that determine the resolution of the mismatch-containing heteroduplexes during CSGE. Like any other mutation scanning technique, CSGE can have limited resolution of two closely linked sequence variations. For specific genes, like BRCA1 and BRCA2 where multiple SNPs are present in the coding sequence, each CSGE shift has to be sequenced to define the exact nature of the sequence change. In conclusion, CSGE scanning provides a powerful, cost-efficient way to scan genes with high sensitivity and specificity.

  18. A method for horizontal polyacrylamide slab gel electrophoresis.

    PubMed

    Bellomy, G R; Record, M T

    1989-01-01

    We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.

  19. Nondenaturing electrophoresis of lipoproteins in agarose and polyacrylamide gradient gels

    SciTech Connect

    Shore, V.G.

    1989-12-19

    The plasma lipoproteins frequently are classified according to density and/or electrophoretic mobility. The lipoprotein classes differ characteristically also in particle size and apolipoprotein composition. Each class is heterogeneous in size and composition as well. Nondenaturing electrophoresis in agarose gels and polyacrylamide gradient gels are complementary analytical methods for classification of lipoproteins and determining distribution profiles of the major classes. In addition, gradient gel electrophoresis (GGE) has a high resolving capability for subfractionating each class according to particle size. Combination of gel electrophoresis with immunoblotting yields information on heterogeneity in apolipoprotein distribution. 14 refs., 6 figs., 3 tabs.

  20. Charge heterogeneity study of a Fc-fusion protein, abatacept, using two-dimensional gel electrophoresis.

    PubMed

    Nebija, D; Noe, C R; Lachmann, B

    2015-08-01

    Medicinal products obtained by recombinant DNA technology are complex molecules and demonstrate a high degree of molecular heterogeneity. Charge heterogeneity and isoform pattern of this class of medicines, are parameters important for their quality, safety, and efficacy. In this study we report the application of two-dimensional gel electrophoresis (2-D electrophoresis) for the quality assessment, identification, charge heterogeneity and isoform pattern study of recombinant protein, CTLA4-Ig (abatacept), which has been selected as an example of the drug class, known as Fc-fusion proteins. In order to achieve an efficient separation of this complex analyte,2-D electrophoresis was optimized employing different experimental conditions regarding the selection of an immobilized pH gradient (IPG), sample pretreatment, presentation and detection procedure. Experimental datadocumented that 2-D electrophoresis is a suitable method for the assessment of identity, purity, structural integrity, isoform pattern and to monitor charge heterogeneity and post-translational glycosylation of the Fc-fusion protein, abatacept.

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

    PubMed

    Slater, Gary W

    2009-06-01

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

  2. Analysis of soybean embryonic axis proteins by two-dimensional gel electrophoresis and mass spectrometry

    USDA-ARS?s Scientific Manuscript database

    A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and subsequent mass spectrometry (MS) for protein identification was applied to establish a proteomic reference map for the soybean embryonic axis. Proteins were extracted from dissecte...

  3. Ocular Proteomics with Emphasis on Two-Dimensional Gel Electrophoresis and Mass Spectrometry

    PubMed Central

    2010-01-01

    The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions. PMID:21406065

  4. RNA conformational changes analyzed by comparative gel electrophoresis.

    PubMed

    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.

  5. Two-dimensional agarose gel electrophoresis of DNA topoisomers.

    PubMed

    Roca, Joaquim

    2009-01-01

    The electrophoretic velocity of a duplex DNA ring is mainly determined by its overall shape. Consequently, DNA topoisomers of opposite supercoiling handedness can have identical gel velocity, and topoisomers highly supercoiled cannot be separated beyond some point. These problems are overcome by two-dimensional agarose gel electrophoresis, which involves two successive electrophoresis steps in one gel slab. The first and second electrophoresis steps are conducted in orthogonal directions with different concentrations of DNA intercalating agents. These compounds alter the overall shape of the DNA and, thereby, change the relative mobility of individual DNA topoisomers.

  6. A Block-matching based technique for the analysis of 2D gel images.

    PubMed

    Freire, Ana; Seoane, José A; Rodríguez, Alvaro; Ruiz-Romero, Cristina; López-Campos, Guillermo; Dorado, Julián

    2010-01-01

    Research at protein level is a useful practice in personalized medicine. More specifically, 2D gel images obtained after electrophoresis process can lead to an accurate diagnosis. Several computational approaches try to help the clinicians to establish the correspondence between pairs of proteins of multiple 2D gel images. Most of them perform the alignment of a patient image referred to a reference image. In this work, an approach based on block-matching techniques is developed. Its main characteristic is that it does not need to perform the whole alignment between two images considering each protein separately. A comparison with other published methods is presented. It can be concluded that this method works over broad range of proteomic images, although they have a high level of difficulty.

  7. Gel Electrophoresis on a Budget to Dye for

    ERIC Educational Resources Information Center

    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…

  8. Gel Electrophoresis on a Budget to Dye for

    ERIC Educational Resources Information Center

    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…

  9. Modified gel preparation for distinct DNA fragment analysis in agarose gel electrophoresis.

    PubMed

    Lee, S V; Bahaman, A R

    2010-08-01

    Agarose gel electrophoresis is the standard method that is used to separate, identify, and purify DNA fragments. However, this method is time-consuming and capable of separating limited range of fragments. A new technique of gel preparation was developed to improve the DNA fragment analysis via electrophoresis.

  10. Pulsed-field gel electrophoresis of bacterial chromosomes.

    PubMed

    Mawer, Julia S P; Leach, David R F

    2013-01-01

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

  11. Gel Electrophoresis--The Easy Way for Students

    ERIC Educational Resources Information Center

    VanRooy, Wilhelmina; Sultana, Khalida

    2010-01-01

    This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

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

    Treesearch

    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....

  13. Gel Electrophoresis--The Easy Way for Students

    ERIC Educational Resources Information Center

    VanRooy, Wilhelmina; Sultana, Khalida

    2010-01-01

    This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

  14. How it all began: a personal history of gel electrophoresis.

    PubMed

    Smithies, Oliver

    2012-01-01

    Arne Tiselius' moving boundary electrophoresis method was still in general use in 1951 when this personal history begins, although zonal electrophoresis with a variety of supporting media (e.g., filter paper or starch grains) was beginning to replace it. This chapter is an account of 10 years of experiments carried out by the author during which molecular sieving gel electrophoresis was developed and common genetic variants of two proteins, haptoglobin and transferrin, were discovered in normal individuals. Most of the figures are images of pages from the author's laboratory notebooks, which are still available, so that some of the excitement of the time and the humorous moments are perhaps apparent. Alkaline gels, acidic gels with and without denaturants, vertical gels, two-dimensional gels, and gels with differences in starch concentration are presented. The subtle details that can be discerned in these various gels played an indispensable role in determining the nature of the change in the haptoglobin gene (Hp) that leads to the polymeric series characteristic of Hp ( 2 ) /Hp ( 2 ) homozygotes. Where possible, the names of scientific friends who made this saga of gel electrophoresis so memorable and enjoyable are gratefully included.

  15. Purification of radiolabeled RNA products using denaturing gel electrophoresis

    PubMed Central

    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 electrophoretic separation of RNA are also discussed. PMID:24510465

  16. Gel Electrophoresis of Gold-DNA Nano-Conjugates

    SciTech Connect

    Pellegrino, T.; Sperling, R.A.; Alivisatos, A.P.; Parak, W.J.

    2006-01-10

    Single stranded DNA of different lengths and different amounts was attached to colloidal phosphine stabilized Au nanoparticles. The resulting conjugates were investigated in detail by a gel electrophoresis study based on 1200 gels. We demonstrate how these experiments help to understand the binding of DNA to Au particles. In particular we compare specific attachment of DNA via gold-thiol bonds with nonspecific adsorption of DNA. The maximum number of DNA molecules that can be bound per particle was determined. We also compare several methods to used gel electrophoresis for investigating the effective diameter of DNA-Au conjugates, such as using a calibration curve of particles with known diameters and Ferguson plots.

  17. A simple gel electrophoresis method for separating polyhedral gold nanoparticles

    NASA Astrophysics Data System (ADS)

    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.

  18. Electric birefrigence imaging of DNA in agarose electrophoresis gels

    SciTech Connect

    Lanan, M.

    1992-01-01

    Electric birefringence imaging (EBI) provides sensitive, non-invasive detection of double-stranded DNA in agarose gels. Quasi-monochromatic, visible light is transmitted through an electrophoresis gel which is placed between plastic film polarizers. A slow-scan video camera equipped with a 12 bit A/D converter records the images. Under electrophoresis running conditions, hydrodynamically-induced gel distortion is shown to be the major source of birefringence for fragments smaller than 23 kbp. The birefringence generated approximates the DNA concentration gradient in the electric field direction. The stress-optic coefficient of 1% agarose gel is measured by mechanical compression and used to evaluate the magnitude of the induced stress on the gel during electrophoresis. Multi-linear regression analysis is used to quantitatively test the model for EBI signals. Birefringence attributed to localized electrokinetic gel distortion and to intrinsic DNA birefringence is studied by fitting ethidium bromide fluorescence profiles to EBI results. Fluorescence polarization imaging is used to assess the influence of localized gel distortion on nucleic acid orientation across a fragment band. It is shown that DNA aligns parallel, on average, with an applied electric field independent of its location within a band. Both EBI sensitivity and quantitation are improved through image processing techniques which separate the DNA Kerr effect and induced electrokinetic distortion contributions. Under standard electrophoresis conditions, detection limits of 8 ng DNA per well are obtained in hydroxyethylated agarose without signal averaging. Maintaining constant gel temperature is shown to improve the quality of the images. Stress patterns in agarose gels during DC and field-inversion gel electrophoresis (FIGE) of nucleic acid fragments of varying sizes are mapped using EBI. In addition, online EBI monitoring during FIGE of megabase pair DNA size standards is demonstrated.

  19. Difference gel electrophoresis (DIGE) using CyDye DIGE fluor minimal dyes.

    PubMed

    Chakravarti, Bulbul; Gallagher, Sean R; Chakravarti, Deb N

    2005-02-01

    One- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) have been widely used for the separation and quantitative estimation of proteins. Following electrophoresis, the gels are stained appropriately to visualize the proteins. Difference gel electrophoresis (DIGE) is a new technique in which different protein samples, individually labeled with specific CyDyes, are combined together followed by electrophoresis and post electrophoretic co-detection and co-analysis on the same gel. CyDye DIGE fluor minimal dyes, which consist of three different CyDyes with different spectral characteristics, have been widely used for such purposes. The technique is highly sensitive with a wide dynamic range for detection of proteins and compatible with state-of-the-art protein identification techniques using mass spectrometry. Although DIGE is mainly used to compare differential expression of various protein samples using 2-D SDS-PAGE, 1-D DIGE also has important applications in quantitative proteomic studies.

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

    PubMed

    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

  1. One-dimensional SDS gel electrophoresis of proteins.

    PubMed

    Gallagher, Sean R

    2006-08-01

    Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full-size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris-tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS-PAGE is a simplified method in which the same buffer is used for both the gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels.

  2. One-dimensional SDS gel electrophoresis of proteins.

    PubMed

    Gallagher, Sean R

    2007-12-01

    Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full-size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris-tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS-PAGE is a simplified method in which the same buffer is used for both the gel and the electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels. (c) 2007 by John Wiley & Sons, Inc.

  3. One-dimensional SDS gel electrophoresis of proteins.

    PubMed

    Gallagher, Sean R

    2006-12-01

    Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full-size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris-tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS-PAGE is a simplified method in which the same buffer is used for both the gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels.

  4. One-dimensional SDS gel electrophoresis of proteins.

    PubMed

    Gallagher, Sean R

    2007-05-01

    Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full-size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris-tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS-PAGE is a simplified method in which the same buffer is used for both the gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels.

  5. Nanoparticle gel electrophoresis: bare charged spheres in polyelectrolyte hydrogels.

    PubMed

    Li, Fei; Hill, Reghan J

    2013-03-15

    Nanoparticle gel electrophoresis has recently emerged as an attractive means of separating and characterizing nanoparticles. Consequently, a theory that accounts for electroosmotic flow in the gel, and coupling of the nanoparticle and hydrogel electrostatics and hydrodynamics, is required, particularly for gels in which the mesh size is comparable to or smaller than the particle radii. Here, we present an electrokinetic model for charged, spherical colloidal particles undergoing electrophoresis in charged (polyelectrolyte) hydrogels: the gel-electrophoresis analogue of Henry's theory for electrophoresis in Newtonian electrolytes. We compare numerically exact solutions of the model with several independent asymptotic approximations, identifying regions in the parameter space where these approximations are accurate or break down. As previously assumed in the literature, Henry's formula, modified by the addition of a constant electroosmotic flow mobility, is accurate only for nanoparticles that are small compared to the hydrogel mesh size. We derived an exact analytical solution of the full model by judiciously modifying the theory of Allison et al. for uncharged gels, drawing on the superposition methodology of Doane et al. to account for hydrogel charge. This furnishes accurate and economical mobility predictions for the entire parameter space. The present model suggests that nanoparticle size separations (with diameters ≲40 nm) are optimal at low ionic strength, with a gel mesh size that is selected according to the particle charging mechanism. For weakly charged particles, optimal size separation is achieved when the Brinkman screening length is matched to the mean particle size.

  6. Inexpensive and safe DNA gel electrophoresis using household materials.

    PubMed

    Ens, S; Olson, A B; Dudley, C; Ross, N D; 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 containers are fitted with aluminum foil electrodes and 9-V batteries to run food-grade agar-agar gels using aquarium pH buffers and then stained with gentian violet. This activity was tested in a high school biology classroom with significantly positive responses on postactivity reflective surveys. The electrophoresis activity addresses several Life Science Content Standard C criteria, including aspects of cell biology, genetics, and evolution. It also can be used to teach aspects of motion and force in the physical science classroom. Copyright © 2012 Wiley Periodicals, Inc.

  7. THERMAL DETECTION OF DNA AND PROTEINS DURING GEL ELECTROPHORESIS

    SciTech Connect

    R. JOHNSTON

    2000-08-01

    This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The goal of this project was to try to detect unstained, untagged, unlabeled DNA bands in real-time during gel electrophoresis using simple thermal measurements. The technical and ES&H advantages to this approach could potentially be quite significant, especially given the extreme importance of gel electrophoresis to a wide variety of practical and research fields. The project was unable to demonstrate sufficient thermal sensitivity to detect DNA bands. It is clear that we still do not understand the gel electrophoresis phenomenon very well. The temperature control techniques developed during the course of this project have other useful applications.

  8. Sample collection system for gel electrophoresis

    DOEpatents

    Olivares, Jose A.; Stark, Peter C.; Dunbar, John M.; Hill, Karen K.; Kuske, Cheryl R.; Roybal, Gustavo

    2004-09-21

    An automatic sample collection system for use with an electrophoretic slab gel system is presented. The collection system can be used with a slab gel have one or more lanes. A detector is used to detect particle bands on the slab gel within a detection zone. Such detectors may use a laser to excite fluorescently labeled particles. The fluorescent light emitted from the excited particles is transmitted to low-level light detection electronics. Upon the detection of a particle of interest within the detection zone, a syringe pump is activated, sending a stream of buffer solution across the lane of the slab gel. The buffer solution collects the sample of interest and carries it through a collection port into a sample collection vial.

  9. A method for easily customizable gradient gel electrophoresis.

    PubMed

    Miller, Andrew J; Roman, Brandon; Norstrom, Eric

    2016-09-15

    Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for routine analysis without the need for specialized mixing equipment. The method allows for easily customizable gradients which can be optimized for specific polypeptide resolution requirements. Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and the time and resources saved with this method in place of traditional gradient mixing, or the purchase of pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Properties of nucleic acid staining dyes used in gel electrophoresis.

    PubMed

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-03-01

    Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen™ and Diamond™ Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.

  11. Enhanced detection of gold nanoparticles in agarose gel electrophoresis.

    PubMed

    Hasenoehrl, Carina; Alexander, Colleen M; Azzarelli, Nicholas N; Dabrowiak, James C

    2012-04-01

    Gel electrophoresis is a powerful tool in gold nanoparticle (AuNP) research. While the technique is sensitive to the size, charge, and shape of particles, its optimal performance requires a relatively large amount of AuNP in the loading wells for visible detection of bands. We here describe a novel and more sensitive method for detecting AuNPs in agarose gels that involves staining the gel with the common organic fluorophore fluorescein, to produce AuNP band intensities that are linear with nanoparticle concentration and almost an order of magnitude larger than those obtained without staining the gel.

  12. Flicker image comparison of 2-D gel images for putative protein identification using the 2DWG meta-database.

    PubMed

    Lemkin, P F; Thornwall, G

    1999-09-01

    With the availability of two-dimensional (2-D) gel electrophoresis databases that have many characterized proteins, it may be possible to compare a researcher's gel images with those in relevant databases. This may lead to the putative identification of unknown protein spots in a researcher's gel with those characterized in a given database, saving the researcher time and money by suggesting monoclonal antibodies to try in confirming these identifications. We have developed two tools to help with this comparison: (1) Flicker, http:/(/)www.lecb.ncifcrf.gov/flicker/, a Java applet program running in the researcher's Web browser, to visually compare their gels against gels on the Internet; and (2) the 2DWG meta-database, http:/(/)www.lecb.ncifcrf.gov/2dwgDB /, a searchable database of locations of 2-D electrophoretic gel images found on the Internet. Recent additions to Flicker allow users to click on a protein spot in a gel that is linked to a federated 2D gel database, such as SWISS-2DPAGE, and have it retrieve a report from that Web database for that protein.

  13. Topological patterns in two-dimensional gel electrophoresis of DNA knots

    PubMed Central

    Michieletto, Davide; Marenduzzo, Davide; Orlandini, Enzo

    2015-01-01

    Gel electrophoresis is a powerful experimental method to probe the topology of DNA and other biopolymers. Although there is a large body of experimental work that allows us to accurately separate different topoisomers of a molecule, a full theoretical understanding of these experiments has not yet been achieved. Here we show that the mobility of DNA knots depends crucially and subtly on the physical properties of the gel and, in particular, on the presence of dangling ends. The topological interactions between these and DNA molecules can be described in terms of an “entanglement number” and yield a nonmonotonic mobility at moderate fields. Consequently, in 2D electrophoresis, gel bands display a characteristic arc pattern; this turns into a straight line when the density of dangling ends vanishes. We also provide a novel framework to accurately predict the shape of such arcs as a function of molecule length and topological complexity, which may be used to inform future experiments. PMID:26351668

  14. Topological patterns in two-dimensional gel electrophoresis of DNA knots.

    PubMed

    Michieletto, Davide; Marenduzzo, Davide; Orlandini, Enzo

    2015-10-06

    Gel electrophoresis is a powerful experimental method to probe the topology of DNA and other biopolymers. Although there is a large body of experimental work that allows us to accurately separate different topoisomers of a molecule, a full theoretical understanding of these experiments has not yet been achieved. Here we show that the mobility of DNA knots depends crucially and subtly on the physical properties of the gel and, in particular, on the presence of dangling ends. The topological interactions between these and DNA molecules can be described in terms of an "entanglement number" and yield a nonmonotonic mobility at moderate fields. Consequently, in 2D electrophoresis, gel bands display a characteristic arc pattern; this turns into a straight line when the density of dangling ends vanishes. We also provide a novel framework to accurately predict the shape of such arcs as a function of molecule length and topological complexity, which may be used to inform future experiments.

  15. One-dimensional SDS gel electrophoresis of proteins.

    PubMed

    Gallagher, Sean R

    2012-04-01

    One-dimensional gel electrophoresis of proteins provides information about the molecular size, amount, and purity of a protein sample. Separated proteins can be recovered from polyacrylamide gels for subsequent characterization by a variety of secondary techniques, such as mass spectrometry to determine post-translational modifications and the amino acid sequence. In addition, one-dimensional electrophoresis is the standard first step in immunoblotting and immunodetection. Protein separations in vertical slab gels are performed in a variety of formats. Most recently, small format minigels are typical due to their ease of use, low relative cost, and ready commercial availability. Larger gels provide more separation area and thus better resolution for complex samples and continue to be in use for specialized analysis. © 2012 by John Wiley & Sons, Inc.

  16. One-dimensional SDS gel electrophoresis of proteins.

    PubMed

    Gallagher, Sean R

    2012-01-01

    One-dimensional gel electrophoresis of proteins provides information about the molecular size, amount, and purity of a protein sample. Separated proteins can be recovered from polyacrylamide gels for subsequent characterization by a variety of secondary techniques, such as mass spectrometry to determine post-translational modifications and the amino acid sequence. In addition, one-dimensional electrophoresis is the standard first step in immunoblotting and immunodetection. Protein separations in vertical slab gels are performed in a variety of formats. Most recently, small format minigels are typical due to their ease of use, low relative cost, and ready commercial availability. Larger gels provide more separation area and thus better resolution for complex samples and continue to be in use for specialized analysis. © 2012 by John Wiley & Sons, Inc.

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

    PubMed

    Murphy, Sandra; Dowling, Paul; Ohlendieck, Kay

    2016-09-09

    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 Chemistry1975, 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.

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

    PubMed Central

    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

  19. Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

    NASA Astrophysics Data System (ADS)

    Ogorzalek Loo, Rachel R.; Hayes, Richard; Yang, Yanan; Hung, Frank; Ramachandran, Prasanna; Kim, Nuri; Gunsalus, Robert; Loo, Joseph A.

    2005-02-01

    Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole-gel trypsin digestion, bottom-up methodology. Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel).

  20. Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis

    SciTech Connect

    Perdew, G.H.; Hollenback, C.E. )

    1990-07-03

    The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-({sup 125}I)-iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BP{sup r}Cl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BP{sup r}Cl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BP{sup r}Cl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is high conserved.

  1. Acrylamide-agarose copolymers: improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis.

    PubMed

    Roncada, Paola; Cretich, Marina; Fortin, Riccardo; Agosti, Susanna; De Franceschi, Lucia; Greppi, Gian Franco; Turrini, Francesco; Carta, Franco; Turri, Stefano; Levi, Marinella; Chiari, Marcella

    2005-06-01

    A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.

  2. Dynamics of DNA molecules under gel electrophoresis

    SciTech Connect

    Kotaka, Tadao, Adachi, Shiro; Shikata, Toshiyuki

    1993-12-31

    Electrophoretic mobilities {mu} of double stranded linear DNAs were examined in agarose gels subjected to a biased sinusoidal field (BSF) that utilizes a sinusoidal field of strength E{sub s} and frequency f superposed on a steady bias field of strength E{sub b}. Under BSF with E{sub s} {much_gt} E{sub b}. DNA fragments with the size M > 20 kbp exhibited peculiar behavior which the authors called a pin down phenomenon in that the {mu} shows a minimum {mu}{sub p} at a particular f{sub p} (pin down frequency) specific to M, C{sub gel} and the field strengths. The dynamics of DNA molecules under such pin-down conditions were examined by direct observation via fluorescence microscopy as well as dynamic electric birefringence.

  3. Fluorescence two-dimensional difference gel electrophoresis for biomaterial applications

    PubMed Central

    McNamara, Laura E.; Dalby, Matthew J.; Riehle, Mathis O.; Burchmore, Richard

    2010-01-01

    Fluorescence two-dimensional difference gel electrophoresis (DiGE) is rapidly becoming established as a powerful technique for the characterization of differences in protein expression levels between two or more conditions. In this review, we consider the application of DiGE—both minimal and saturation labelling—to biomaterials research, considering the challenges and rewards of this approach. PMID:19570793

  4. Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates

    USDA-ARS?s Scientific Manuscript database

    Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by th...

  5. The Gel Electrophoresis Markup Language (GelML) from the Proteomics Standards Initiative

    PubMed Central

    Gibson, Frank; Hoogland, Christine; Martinez-Bartolomé, Salvador; Medina-Aunon, J. Alberto; Albar, Juan Pablo; Babnigg, Gyorgy; Wipat, Anil; Hermjakob, Henning; Almeida, Jonas S; Stanislaus, Romesh; Paton, Norman W; Jones, Andrew R

    2011-01-01

    The Human Proteome Organisation’s Proteomics Standards Initiative (HUPO-PSI) has developed the GelML data exchange format for representing gel electrophoresis experiments performed in proteomics investigations. The format closely follows the reporting guidelines for gel electrophoresis, which are part of the Minimum Information About a Proteomics Experiment (MIAPE) set of modules. GelML supports the capture of metadata (such as experimental protocols) and data (such as gel images) resulting from gel electrophoresis so that laboratories can be compliant with the MIAPE Gel Electrophoresis guidelines, while allowing such data sets to be exchanged or downloaded from public repositories. The format is sufficiently flexible to capture data from a broad range of experimental processes, and complements other PSI formats for mass spectrometry data and the results of protein and peptide identifications to capture entire gel-based proteome workflows. GelML has resulted from the open standardisation process of PSI consisting of both public consultation and anonymous review of the specifications. PMID:20677327

  6. The new horizon in 2D electrophoresis: new technology to increase resolution and sensitivity.

    PubMed

    Moche, Martin; Albrecht, Dirk; Maaß, Sandra; Hecker, Michael; Westermeier, Reiner; Büttner, Knut

    2013-06-01

    A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of precast polyacrylamide gels on film-backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected.

  7. Two-dimensional gel electrophoresis: vertical isoelectric focusing.

    PubMed

    Dorri, Yaser

    2012-01-01

    Two-dimensional gel electrophoresis (2-DE) is one of the most powerful tools for separating proteins based on their size and charge. 2-DE is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical SDS-PAGE apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and, after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

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

    PubMed

    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.

  9. Purification of coated vesicles by agarose gel electrophoresis

    PubMed Central

    1981-01-01

    We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. Coated vesicles from three different cell types have different mobilities. In each case, however, all of the major polypeptides previously attributed to coated vesicles comigrate with the now homogeneous particles, even though a powerful ATPase activity is completely removed. PMID:6265464

  10. Separation of glutathione transferase subunits from Proteus vulgaris by two-dimensional gel electrophoresis.

    PubMed

    Hong, Giaming; Chien, Yi-Chih; Chien, Cheng-I

    2003-10-01

    Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.

  11. Two-dimensional agarose gel electrophoresis for analysis of DNA replication.

    PubMed

    Villwock, Sandra K; Aparicio, Oscar M

    2014-01-01

    The initiation, elongation, and termination of DNA replication are each associated with distinct, nonlinear DNA structures that can be resolved and identified by two-dimensional (2D) agarose gel electrophoresis. This method involves: isolation of genomic DNA while preserving fragile replication structures, digestion of the DNA with a restriction enzyme, separation of DNA by size and shape through two distinct stages of agarose gel electrophoresis, and Southern blotting to probe for the specific sequence(s) of interest. The method has been most commonly used to determine the activity level of putative replication origin-containing sequences, and has also been used to analyze replication timing, fork progression, fork pausing, fork stalling and collapse, termination, and recombinational repair.

  12. Thermally reversible gels in electrophoresis. I - Matrix characterization

    NASA Technical Reports Server (NTRS)

    Righetti, Pier Giorgio; Snyder, Robert S.

    1988-01-01

    Two series of thermally reversible hydrogen-bonded gels have been characterized: (5 pct) PVA-(4 pct) PEG and (5 pct) PVA-(0.04 pct) borate gels. They both have extremely low melting points (16-17 C) and could be of potential interest for recovery of proteins after preparative electrophoresis. The PVA-borate gels can be exploited in the pH range 7-11 by progressively increasing the borate content in the pH interval 8 to 7 and concomitantly decreasing the borate levels in the pH zone 8 to 11. It is hypothesized that the low melting point of these gels is due to the fact that they are sparingly and sparsely hydrogen bonded along the PVA chain: on the average, 1 OH group out of 3 or 4 OH groups in the PVA polymer should be engaged in H-bond formation.

  13. Thermally reversible gels in electrophoresis. I - Matrix characterization

    NASA Technical Reports Server (NTRS)

    Righetti, Pier Giorgio; Snyder, Robert S.

    1988-01-01

    Two series of thermally reversible hydrogen-bonded gels have been characterized: (5 pct) PVA-(4 pct) PEG and (5 pct) PVA-(0.04 pct) borate gels. They both have extremely low melting points (16-17 C) and could be of potential interest for recovery of proteins after preparative electrophoresis. The PVA-borate gels can be exploited in the pH range 7-11 by progressively increasing the borate content in the pH interval 8 to 7 and concomitantly decreasing the borate levels in the pH zone 8 to 11. It is hypothesized that the low melting point of these gels is due to the fact that they are sparingly and sparsely hydrogen bonded along the PVA chain: on the average, 1 OH group out of 3 or 4 OH groups in the PVA polymer should be engaged in H-bond formation.

  14. Pulsed field gel electrophoresis on frozen tumour tissue sections.

    PubMed Central

    Boultwood, J.; Kaklamanis, L.; Gatter, K. C.; Wainscoat, J. S.

    1992-01-01

    The application of pulsed field gel electrophoresis (PFGE) to the molecular genetic analysis of solid tumours has been restricted by the requirement for whole single cells as a DNA source. A simple technique which allows for the direct analysis of histologically characterised solid tumour material by pulsed field gel electrophoresis was developed. Single frozen tissue sections obtained from colonic carcinoma specimens were embedded without further manipulation in molten, low melting temperature agarose. The tumour DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is yielded by this method to obtain a hybridisation signal with a single copy probe. Histological examination of adjacent tissue sections may also be carried out, permitting correlation between molecular analysis and tumour histology. Images PMID:1401187

  15. Blood grouping based on PCR methods and agarose gel electrophoresis.

    PubMed

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  16. Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis

    PubMed Central

    2014-01-01

    Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional- polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunological tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodological advances and new applications. PMID:24735559

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

    PubMed

    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.

  18. Two-dimensional difference gel electrophoresis applied for analytical proteomics: fundamentals and applications to the study of plant proteomics.

    PubMed

    Arruda, Sandra Cristina Capaldi; Barbosa, Herbert de Sousa; Azevedo, Ricardo Antunes; Arruda, Marco Aurélio Zezzi

    2011-10-21

    The present review reports the principles, fundamentals and some applications of two-dimensional difference gel electrophoresis for analytical proteomics based on plant proteome analysis, also emphasizing some advantages of 2-D DIGE over 2-D PAGE techniques. Some fluorescent protein labeling reagents, methods of protein labeling, models of 2-D DIGE experiments, and some limitations of this technique are presented and discussed in terms of 2-D DIGE plant proteomes. Finally, some practical applications of this technique are pointed out, emphasizing its potentialities in plant proteomics.

  19. Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

    PubMed Central

    Childs, J D; Birnboim, H C

    1975-01-01

    A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses. Images PMID:240037

  20. A Bayesian model for classifying all differentially expressed proteins simultaneously in 2D PAGE gels.

    PubMed

    Wu, Steven H; Black, Michael A; North, Robyn A; Rodrigo, Allen G

    2012-06-19

    Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is commonly used to identify differentially expressed proteins under two or more experimental or observational conditions. Wu et al (2009) developed a univariate probabilistic model which was used to identify differential expression between Case and Control groups, by applying a Likelihood Ratio Test (LRT) to each protein on a 2D PAGE. In contrast to commonly used statistical approaches, this model takes into account the two possible causes of missing values in 2D PAGE: either (1) the non-expression of a protein; or (2) a level of expression that falls below the limit of detection. We develop a global Bayesian model which extends the previously described model. Unlike the univariate approach, the model reported here is able treat all differentially expressed proteins simultaneously. Whereas each protein is modelled by the univariate likelihood function previously described, several global distributions are used to model the underlying relationship between the parameters associated with individual proteins. These global distributions are able to combine information from each protein to give more accurate estimates of the true parameters. In our implementation of the procedure, all parameters are recovered by Markov chain Monte Carlo (MCMC) integration. The 95% highest posterior density (HPD) intervals for the marginal posterior distributions are used to determine whether differences in protein expression are due to differences in mean expression intensities, and/or differences in the probabilities of expression. Simulation analyses showed that the global model is able to accurately recover the underlying global distributions, and identify more differentially expressed proteins than the simple application of a LRT. Additionally, simulations also indicate that the probability of incorrectly identifying a protein as differentially expressed (i.e., the False Discovery Rate) is very low. The source code is

  1. Molecular transport in collagenous tissues measured by gel electrophoresis.

    PubMed

    Hunckler, Michael D; Tilley, Jennifer M R; Roeder, Ryan K

    2015-11-26

    Molecular transport in tissues is important for drug delivery, nutrient supply, waste removal, cell signaling, and detecting tissue degeneration. Therefore, the objective of this study was to investigate gel electrophoresis as a simple method to measure molecular transport in collagenous tissues. The electrophoretic mobility of charged molecules in tissue samples was measured from relative differences in the velocity of a cationic dye passing through an agarose gel in the absence and presence of a tissue section embedded within the gel. Differences in electrophoretic mobility were measured for the transport of a molecule through different tissues and tissue anisotropy, or the transport of different sized molecules through the same tissue. Tissue samples included tendon and fibrocartilage from the proximal (tensile) and distal (compressive) regions of the bovine flexor tendon, respectively, and bovine articular cartilage. The measured electrophoretic mobility was greatest in the compressive region of the tendon (fibrocartilage), followed by the tensile region of tendon, and lowest in articular cartilage, reflecting differences in the composition and organization of the tissues. The anisotropy of tendon was measured by greater electrophoretic mobility parallel compared with perpendicular to the predominate collagen fiber orientation. Electrophoretic mobility also decreased with increased molecular size, as expected. Therefore, the results of this study suggest that gel electrophoresis may be a useful method to measure differences in molecular transport within various tissues, including the effects of tissue type, tissue anisotropy, and molecular size.

  2. Higher sensitivity of capillary electrophoresis in detecting hemoglobin A2'compared to traditional gel electrophoresis.

    PubMed

    Oleske, Deanna Alicia; Huang, Richard Sheng Poe; Dasgupta, Amitava; Nguyen, Andy; Wahed, Amer

    2014-01-01

    HbA2' (also called Hb B2) is the most common delta-globin chain defect and is reported to occur in 1-2% of the African American population. The major clinical significance of HbA2' is that the failure to detect it might lead to an underestimation of the total HbA2, leading to failure to diagnose β-thalassemia minor. In order to diagnose β-thalassemia minor, both HbA2 and HbA2' levels must be combined.Hb A2' accounts for a small percentage (1-2%) of the total hemoglobin in heterozygotes. It is difficult to detect this small amount by traditional gel electrophoresis. Using HPLC Hb A2' is easily detected as it produces a minor peak in the S window. Other conditions which might interfere with detection of HbA2' by HPLC include Hb S trait or Hb SS disease (Hb A2' hidden in the S peak), transfused Hb SS (Hb S peak may be very small), Hb C trait or Hb CC disease (glycosylated Hb C elutes in the S window), and Hb G (Hb G2 elutes in the S window). All of the above conditions, including Hb A2', occur most commonly in the same ethnic group (African American). We reviewed 654 consecutive cases over a period of three months for the presence of Hb A2' in our laboratory where capillary electrophoresis is used as the primary diagnostic tool. We detected seven cases (1.07 %) of HbA2'. In contrast, we did not detect any HbA2' using conventional gel electrophoresis in the last one year (2,580 cases). Although in none of the seven cases the sum of Hb A2 and Hb A2' exceeded 3.5%, we believe that capillary electrophoresis allows for a better detection of Hb A2' than gel electrophoresis and HPLC.

  3. Graphitic carbon nitride embedded hydrogels for enhanced gel electrophoresis.

    PubMed

    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.

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

    PubMed

    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.

  5. Nanostructured Copolymer Gels for dsDNA Separation by Capillary Electrophoresis

    PubMed Central

    Wan, Fen; Zhang, Jun; Lau, Angela; Tan, Sarah; Burger, Christian; Chu, Benjamin

    2010-01-01

    Pluronics copolymers are triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) and are able to form many different ordered nanostructures at appropriate polymer concentrations and temperatures in selective solvents. These nano-structured ‘gels’ showed desirable criteria when used as DNA separation media, especially in microchip electrophoresis, including dynamic coating ability and viscosity switchable property. A ternary system of F127 (E99P69E99)/TBE buffer/1-butanol was selected as a model system to test the sieving performance of different nanostructures in separating dsDNA by capillary electrophoresis. The lattice structures were determined by small-angle x-ray scattering with quasi-lattice crystal parameters being calculated according to the x-ray scattering data. Viscosity measurements showed the sol-gel transition phenomena. In addition to the cubic structure, successful electrophoretic separation of dsDNA in 2-D hexagonal packed cylinders was achieved. Results showed that without further optimization, ΦX174 DNA-Hae III digest was well separated within 15 minutes in a 7-cm separation channel, by using F127/TBE/1-butanol gel with a 2-D hexagonal structure. A mechanism for DNA separations by those gels with both hydrophilic and hydrophobic domains is discussed. PMID:19053068

  6. Gel and free solution electrophoresis of variably charged polymers.

    PubMed

    Hoagland, D A; Smisek, D L; Chen, D Y

    1996-06-01

    To assess the role of charge density on polyelectrolyte mobility, both gel and free solution electrophoresis experiments are performed on poly(acrylic acid) and acrylic acid/acrylamide copolymers. Control of charge density for poly-(acrylic acid) is achieved through solution pH, while control for acrylic acid/ acrylamide copolymers is obtained through chain composition. In either approach, the effective fraction of charged repeat units can be varied from 0 to 100% without a major interruption of solvent quality. Polyelectrolyte mobility in the presence of a monovalent counterion is observed to rise linearly with charge density when this density is low. A transition to charge density independence then occurs over a surprisingly narrow window of charge density. For vinyl polymers of the sort examined here, the transition occurs when 35-40% of the repeat units are charged. These observations are qualitatively consistent with the free solution electrophoresis model proposed by Manning and several previous data sets. An unexpected overlap of normalized gel and free solution data reveals that the charge density exerts a comparable influence in either environment. Results from the present study help define the experimental conditions in which electrophoresis can provide polymer separation by charge density and those in which the method can provide polymer separation by molecular weight.

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

    SciTech Connect

    Xu, Aoshuang

    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.

  8. A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet.

    PubMed

    Mollenkopf, H J; Jungblut, P R; Raupach, B; Mattow, J; Lamer, S; Zimny-Arndt, U; Schaible, U E; Kaufmann, S H

    1999-08-01

    Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.

  9. A continuous acetic acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines.

    PubMed

    Clements, R L

    1988-02-01

    A polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate-lactic acid system (using 12% acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.

  10. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry.

    USDA-ARS?s Scientific Manuscript database

    Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of...

  11. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

    PubMed Central

    Stellwagen, Nancy C.

    2009-01-01

    This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due primarily to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 ± 0.01) × 10-4 cm2/Vs in 40 mM Tris-acetate-EDTA buffer at 20°C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration. PMID:19517510

  12. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution.

    PubMed

    Stellwagen, Nancy C

    2009-06-01

    This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are primarily due to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 +/- 0.01) x 10(-4) cm2/V s in 40 mM Tris-acetate-EDTA buffer at 20 degrees C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.

  13. Two-dimensional gel electrophoresis in bacterial proteomics.

    PubMed

    Curreem, Shirly O T; Watt, Rory M; Lau, Susanna K P; Woo, Patrick C Y

    2012-05-01

    Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.

  14. pK-matched running buffers for gel electrophoresis.

    PubMed

    Liu, Q; Li, X; Sommer, S S

    1999-05-15

    Electrophoresis through agarose and polyacrylamide-type gels is the standard method to separate, identify, and purify nucleic acids. Properties of electrophoresis buffers such as pH, ionic strength, and composition affect performance. The buffers in use contain a weak acid or weak base buffered by a compound with a dissimilar pK. Herein, three pK-matched buffers were developed, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25 degrees C (within 0.3 pK units): (i) Ethanolamine/Capso, pH 9.6; (ii) triethanolamine/Tricine, pH 7.9; and (iii) Bis-Tris/Aces, pH 6.7. On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/acetate, pH 8.0) or TBE (Tris/borate, pH 8.3) buffers. The buffers were also tested in 7 M urea denaturing LongRanger sequencing gels and in nondenaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions (up to 200x), and lower current per unit voltage, resulting in less heat generation. Copyright 1999 Academic Press.

  15. System for loading slab-gel holders for electrophoresis separation

    DOEpatents

    Anderson, Norman G.; Anderson, Norman L.

    1979-01-01

    A slab-gel loading system includes a prismatic chamber for filling a plurality of slab-gel holders simultaneously. Each slab-gel holder comprises a pair of spaced apart plates defining an intermediate volume for gel containment. The holders are vertically positioned in the chamber with their major surfaces parallel to the chamber end walls. A liquid inlet is provided at the corner between the bottom and a side wall of the chamber for distributing a polymerizable monomer solution or a coagulable colloidal solution into each of the holders. The chamber is rotatably supported so that filling can begin with the corner having the liquid inlet directed downwardly such that the solution is gently funneled upwardly, without mixing, along the diverging side and bottom surfaces. As filling proceeds, the chamber is gradually rotated to position the bottom wall in a horizontal mode. The liquid filling means includes a plastic envelope with a septum dividing it into two compartments for intermixing two solutions of different density and thereby providing a liquid flow having a density gradient. The resulting gels have a density gradient between opposite edges for subsequent use in electrophoresis separations.

  16. Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms.

    PubMed

    Niyomploy, Ploypat; Srisomsap, Chantragan; Chokchaichamnankit, Daranee; Vinayavekhin, Nawaporn; Karnchanatat, Aphichart; Sangvanich, Polkit

    2014-03-01

    Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future.

  17. Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis.

    PubMed

    Fivaz, M; Vilbois, F; Pasquali, C; van der Goot, F G

    2000-10-01

    The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.

  18. Electrophoresis and orientation of F-actin in agarose gels.

    PubMed

    Borejdo, J; Ortega, H

    1989-08-01

    F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase activity of HMM. The mobility of F-actin increased with the rise in pH. Acto-S-1 complex was also able to migrate in agarose at basic pH, but at a lower rate than F-actin alone. The orientation of fluorescein labeled F-actin and of fluorescein labeled S-1 which formed rigor bonds with F-actin was measured during the electrophoresis by the fluorescence detected linear dichroism method. The former showed little orientation, probably because the dye was mobile on the surface of actin, but we were able to measure the orientation of the absorption dipole of the dye bound to S-1 which was attached to F-actin, and found that it assumed an orientation largely parallel to the direction of the electric field. These results show that actin can migrate in agarose gels in the F form and that it is oriented during the electrophoresis.

  19. Two-Dimensional Gel Electrophoresis: A Reference Protocol.

    PubMed

    Saia-Cereda, Veronica M; Aquino, Adriano; Guest, Paul C; Martins-de-Souza, Daniel

    2017-01-01

    Two-dimensional gel electrophoresis (2DE) has been a mainstay of proteomic techniques for more than four decades. It was even in use for several years before the term proteomics was actually coined in the early 1990s. Over this time, it has been used in the study of many diseases including cancer, diabetes, heart disease, and psychiatric disorders through the proteomic analysis of body fluids and tissues. This chapter presents a general protocol which can be applied in the study of biological samples such as blood serum or plasma and multiple tissues including the brain.

  20. Challenges of glycoprotein analysis by microchip capillary gel electrophoresis.

    PubMed

    Engel, Nicole; Weiss, Victor U; Wenz, Christian; Rüfer, Andreas; Kratzmeier, Martin; Glück, Susanne; Marchetti-Deschmann, Martina; Allmaier, Günter

    2015-08-01

    Glycosylations severely influence a protein's biological and physicochemical properties. Five exemplary proteins with varying glycan moieties were chosen to establish molecular weight (MW) determination (sizing), quantitation, and sensitivity of detection for microchip capillary gel electrophoresis (MCGE). Although sizing showed increasing deviations from literature values (SDS-PAGE or MALDI-MS) with a concomitant higher degree of analyte glycosylation, the reproducibility of MW determination and accuracy of quantitation with high sensitivity and reliability were demonstrated. Additionally, speed of analysis together with the low level of analyte consumption render MCGE attractive as an alternative to conventional SDS-PAGE.

  1. Electrophoresis for genotyping: temporal thermal gradient gel electrophoresis for profiling of oligonucleotide dissociation.

    PubMed Central

    Day, I N; O'Dell, S D; Cash, I D; Humphries, S E; Weavind, G P

    1995-01-01

    Traditional use of an oligonucleotide probe to determine genotype depends on perfect base pairing to a single-stranded target which is stable to a higher temperature than when imperfect binding occurs due to a mismatch in the target sequence. Bound oligonucleotide is detected at a predetermined single temperature 'snapshot' of the melting profile, allowing the distinction of perfect from imperfect base pairing. In heterozygotes, the presence of the alternative sequence must be verified with a second oligonucleotide complementary to the variant. Here we describe a system of real-time variable temperature electrophoresis during which the oligonucleotide dissociates from its target. In 20% polyacrylamide the target strand has minimal mobility and released oligonucleotide migrates extremely quickly so that the 'freed' rather than the 'bound' is displayed. The full profile of oligonucleotide dissociation during gel electrophoresis is represented along the gel track, and a single oligonucleotide is sufficient to confirm heterozygosity, since the profile displays two separate peaks. Resolution is great, with use of short track lengths enabling analysis of dense arrays of samples. Each gel track can contain a different target or oligonucleotide and the temperature gradient can accommodate oligonucleotides of different melting temperatures. This provides a convenient system to examine the interaction of many different oligonucleotides and target sequences simultaneously and requires no prior knowledge of the mutant sequence(s) nor of oligonucleotide melting temperatures. The application of the technique is described for screening of a hotspot for mutations in the LDL receptor gene in patients with familial hypercholesterolaemia. Images PMID:7630718

  2. Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis.

    PubMed

    Huettel, R N; Dickson, D W; Kaplan, D T

    1983-07-01

    Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes.

  3. Separation of radiolabelled glycosaminoglycan oligosaccharides by polyacrylamide-gel electrophoresis.

    PubMed

    Hampson, I N; Gallagher, J T

    1984-08-01

    Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.

  4. GELBANK : A database of annotated two-dimensional gel electrophoresis patterns of biological systems with completed genomes.

    SciTech Connect

    Babnigg, G.; Giometti, C. S.; Biosciences Division

    2004-01-01

    GELBANK is a publicly available database of two-dimensional gel electrophoresis (2DE) gel patterns of proteomes from organisms with known genome information (available at and ftp://bioinformatics.anl.gov/gelbank/). Currently it includes 131 completed, mostly microbial proteomes available from the National Center for Biotechnology Information. A web interface allows the upload of 2D gel patterns and their annotation for registered users. The images are organized by species, tissue type, separation method, sample type and staining method. The database can be queried based on protein or 2DE-pattern attributes. A web interface allows registered users to assign molecular weight and pH gradient profiles to their own 2D gel patterns as well as to link protein identifications to a given spot on the pattern. The website presents all of the submitted 2D gel patterns where the end-user can dynamically display the images or parts of images along with molecular weight, pH profile information and linked protein identification. A collection of images can be selected for the creation of animations from which the user can select sub-regions of interest and unlimited 2D gel patterns for visualization. The website currently presents 233 identifications for 81 gel patterns for Homo sapiens, Methanococcus jannaschii, Pyro coccus furiosus, Shewanella oneidensis, Escherichia coli and Deinococcus radiodurans.

  5. Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis.

    PubMed

    Schrodt, Caroline; McHugh, Erin E; Gawinowicz, Mary Ann; Dupont, Herbert L; Brown, Eric L

    2013-01-01

    Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens.

  6. Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis

    PubMed Central

    Schrodt, Caroline; McHugh, Erin E.; Gawinowicz, Mary Ann; DuPont, Herbert L.; Brown, Eric L.

    2013-01-01

    Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens. PMID:23922656

  7. Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis.

    PubMed

    Yokoyama, Eiji; Kishida, Kazunori; Uchimura, Masako; Ichinohe, Sadato

    2006-06-01

    Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.

  8. Dye removal, catalytic activity and 2D crystallization of chloroplast H(+)-ATP synthase purified by blue native electrophoresis.

    PubMed

    Poetsch, A; Neff, D; Seelert, H; Schägger, H; Dencher, N A

    2000-06-01

    The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.

  9. Optimal processing for gel electrophoresis images: Applying Monte Carlo Tree Search in GelApp.

    PubMed

    Nguyen, Phi-Vu; Ghezal, Ali; Hsueh, Ya-Chih; Boudier, Thomas; Gan, Samuel Ken-En; Lee, Hwee Kuan

    2016-08-01

    In biomedical research, gel band size estimation in electrophoresis analysis is a routine process. To facilitate and automate this process, numerous software have been released, notably the GelApp mobile app. However, the band detection accuracy is limited due to a band detection algorithm that cannot adapt to the variations in input images. To address this, we used the Monte Carlo Tree Search with Upper Confidence Bound (MCTS-UCB) method to efficiently search for optimal image processing pipelines for the band detection task, thereby improving the segmentation algorithm. Incorporating this into GelApp, we report a significant enhancement of gel band detection accuracy by 55.9 ± 2.0% for protein polyacrylamide gels, and 35.9 ± 2.5% for DNA SYBR green agarose gels. This implementation is a proof-of-concept in demonstrating MCTS-UCB as a strategy to optimize general image segmentation. The improved version of GelApp-GelApp 2.0-is freely available on both Google Play Store (for Android platform), and Apple App Store (for iOS platform).

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

    PubMed

    Nałęcz, Dorota; Dziuba, Marta; Szerszunowicz, Iwona

    2017-01-01

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

  11. Red wine proteins: two dimensional (2-D) electrophoresis and mass spectrometry analysis.

    PubMed

    Mainente, Federica; Zoccatelli, Gianni; Lorenzini, Marilinda; Cecconi, Daniela; Vincenzi, Simone; Rizzi, Corrado; Simonato, Barbara

    2014-12-01

    The aim of the present study was to optimize protein extraction from red wine (cv. Cabernet) in order to obtain a separation by two-dimensional electrophoresis (2-DE) compatible with mass spectrometry identification. Proteins were denatured by sodium dodecyl-sulphate (SDS) and precipitated as potassium salts. The potassium-DS (KDS) protein complexes obtained were treated with different solutions in order to remove the detergent. Proteins were solubilized with different buffers and separated by different electrophoretic approaches [native, urea, acid urea PAGEs and isoelectric focusing (IEF)] as the first-dimension (1-DE). The best 2D separation was achieved by using 10% saccharose in the DS removal step, and 6-cyclohexylhexyl β-d-maltoside detergent in the solubilisation buffer combined with the IEF approach. Several well focalized protein spots were obtained and analyzed through mass-spectrometry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Sarma, Annamraju D; Oehrle, Nathan W; Emerich, David W

    2008-08-15

    Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 degrees C, it was carried out for 2 to 3h at -80 degrees C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 degrees C. The applicability of the method was demonstrated using several soybean tissues.

  13. Heterogeneity of liver alcohol dehydrogenase on starch-gel electrophoresis

    PubMed Central

    McKinley-McKee, J. S.; Moss, D. W.

    1965-01-01

    1. Purified horse-liver alcohol dehydrogenase is heterogeneous on starch-gel electrophoresis in several buffer systems. 2. The electrophoretic pattern is altered by the addition to the buffers of oxidized or reduced coenzymes, isobutyramide, metal ions or metal-chelating agents. 3. The effect of coenzymes on the pattern suggests that the major cause of the observed heterogeneity is not the existence of isoenzymes, but the presence in the enzyme preparations of coenzyme–enzyme complexes or complexes with other nucleotides similar to, but less reactive than, the coenzymes. 4. Metal ions and chelating agents influence the electrophoretic separation by partial denaturation and inactivation of the enzyme. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:4285890

  14. Analyzing genetic diversity in conifers...isozyme resolution by starch gel electrophoresis

    Treesearch

    M. Thompson Conkle

    1972-01-01

    Enzymes in forest tree materials can be resolved by starch gel electrophoresis. A gel slab is prepared in a mold assembled from glass and plastic. Wicks containing an aqueous extract of macerated plant material are inserted in the gel and processed. The gel is sliced, stained, examined, and photographed. Isozyme bands produced by differential migration of enzymes...

  15. Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels.

    PubMed

    Muratsubaki, Haruhiro; Satake, Kaoru; Yamamoto, Yasuhisa; Enomoto, Keiichiro

    2002-08-15

    Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.

  16. Analysis of strains of Campylobacter fetus by pulsed-field gel electrophoresis.

    PubMed Central

    Fujita, M; Fujimoto, S; Morooka, T; Amako, K

    1995-01-01

    Campylobacter fetus chromosomal DNA from 21 strains was analyzed by pulsed-field gel electrophoresis. The fingerprint patterns generated with SmaI and SalI were distinctive. Using the profiles obtained by pulsed-field gel electrophoresis, we established the phylogenetic dendrogram of C. fetus to identify the genetic relationship of the strains. PMID:7650215

  17. DNA Length Ranges Exhibiting Distinct Separation Mechanisms in Gel Electrophoresis

    NASA Astrophysics Data System (ADS)

    Beheshti, A.; van Winkle, D. H.; Rill, R. L.

    2003-03-01

    Electrophoresis was performed on double stranded DNA ranging from 200 to 194,000 bp in agarose gel concentrations from 0.4% - 1.3%. The electric field was varied from 0.62 to 6.21 V/cm. A wide range of electric fields and gel concentrations were used to study how the new interpolation equation, frac1μ(L) = frac1μL - (frac1μL - frac1μ_s)e^-L/γ (where μ_L, μ_s, and γ are independent free fitting parameters), helps to distinguish among different mechanisms of molecular transport. This exponential relation fits well when there is a smooth transition from Ogston sieving to reptation. These transitions are distinguished by so-called ``reptation plots" (plotting 3μ L/μ_rc vs. L) (J. Rousseau, G. Drouin, and G. W. Slater, Phys Rev Lett. 1997, 79, 1945-1948). Fits deviate from the data more than two characteristic trends are observed in the reptation plots. The failure of the fits to follow the data appears to be a consequence of another separation mechanism, ``entropic trapping," occurring between the sieving and reptation regimes. The boundaries between length and field ranges where different separation mechanisms dominate are extracted from reptation plots of the best fits and the data. ``Phase diagrams" expressing these boundaries are derived.

  18. Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis.

    PubMed

    Luo, Xiaoting; Wu, Jinzi; Jin, Zhen; Yan, Liang-Jun

    2017-02-02

    Gradient blue native polyacrylamide gel electrophoresis (BN-PAGE) is a well established and widely used technique for activity analysis of high-molecular-weight proteins, protein complexes, and protein-protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non-gradient BN-PAGE. The procedure, similar to that of non-gradient SDS-PAGE, is simple because there is no expensive gradient maker involved. The non-gradient BN-PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non-gradient BN-PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  19. Characterization of Canine Mycoplasmas by Polyacrylamide Gel Electrophoresis and Immunodiffusion

    PubMed Central

    Armstrong, D.; Yu, B.

    1970-01-01

    Canine mycoplasmas which had been characterized by biological and serological methods were further studied by using polyacrylamide gel electrophoresis (PGE) and double diffusion in agar gel. The three dog mycoplasmas previously characterized, Mycoplasma canis, M. maculosum, and M. spumans showed distinctive patterns by PGE. Five additional representative isolates from dogs had been characterized serologically and biologically into three new groups, A, C, and D. An additional mycoplasma (group B) was indistinguishable from M. canis by growth inhibition and PGE but was more broadly reactive with field isolates serologically. The group A organisms were distinctive in pattern and similar to those studied by Razin and Rottem, tentatively designated M. edwardii. The group C organisms were represented by two isolates which were similar by fluorescent-antibody studies but different by growth inhibition tests. These two isolates were also different from each other by PGE. The group D serotypes were also distinctive by PGE from all other dog mycoplasmas tested. It was found, during these studies, that two different mycoplasmas showed different PGE patterns at different intervals during incubation. Immunodiffusion studies showed a relationship among all the canine mycoplasmas, and bands of nonidentity between the two group C mycoplasmas were demonstrated. Images PMID:4990761

  20. Two-dimensional blue native polyacrylamide gel electrophoresis.

    PubMed

    Schamel, Wolfgang W A

    2008-03-01

    Multiprotein complexes play crucial roles in nearly all cell biological processes. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a powerful method to study these complexes. It is a native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. It has a higher resolution than gel filtration or sucrose density ultracentrifugation and can be used for protein complexes from 10 kDa to 10 MDa. If a second-dimension SDS-PAGE is applied (two-dimensional BN/SDS-PAGE), the size, subunit composition, and relative abundance of the different multiprotein complexes can be studied. In recent years, there has been a large increase in the number of publications where BN-PAGE was used to study protein-protein interactions. Here, we give detailed protocols for the separation of multiprotein complexes by two-dimensional BN/SDS-PAGE and for a related technique to determine the stoichiometry of these complexes. (c) 2008 by John Wiley & Sons, Inc.

  1. Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method ▿ §

    PubMed Central

    Agafonov, Dmitry E.; Deckert, Jochen; Wolf, Elmar; Odenwälder, Peter; Bessonov, Sergey; Will, Cindy L.; Urlaub, Henning; Lührmann, Reinhard

    2011-01-01

    More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, Bact, and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, Bact, and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing. PMID:21536652

  2. Development of bufferless gel electrophoresis chip for easy preparation and rapid DNA separation.

    PubMed

    Oleksandrov, Sergiy; Aman, Abdurazak; Lim, Wanyoung; Kim, Younghee; Bae, Nam Ho; Lee, Kyoung G; Lee, Seok Jae; Park, Sungsu

    2017-09-27

    This work presents a handy, fast, and compact bufferless gel electrophoresis chip (BGEC), which consists of precast agarose gel confined in a disposable plastic body with electrodes. It does not require large volumes of buffer to fill reservoirs, or the process of immersing the gel in the buffer. It withstands voltages up to 28.4 V/cm, thereby allowing DNA separation within 10 minutes with a similar separation capability to the standard gel electrophoresis. The results suggest that our BGEC is highly suitable for in situ gel electrophoresis in forensic, epidemiological settings and crime scenes where standard gel electrophoresis equipment cannot be brought in while quick results are needed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

    SciTech Connect

    Lacks, S.A.; Springhorn, S.S.

    1980-08-10

    A number of enzymes, including amylases, dehydrogenases, and proteases, were shown to be renaturable after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Enzyme activity was detected in situ by action on substrates introduced into the gel and subsequent staining of either the product or unreacted substrate. Enzymes appeared to recover activity as soon as the detergent diffused out of the gel. Renatured enzymes were retained in gels after electrophoresis longer than native enzymes which had been subjected to electrophoresis in the absence of detergent. Re-electrophoresis of the renatured enzymes showed that part of the retained activity was physically anchored to the gel, possibly by the folding of polypeptides around the gel matrix as the enzymes were renatured.

  4. Tear film proteins deposited on high water content contact lenses identified with two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Nielsen, Kim; Vorum, Henrik; Ehlers, Niels; Aagaard, Nicolaj; Hjortdal, Jesper; Honoré, Bent

    2015-11-01

    Tear film proteins adhere to the surface of contact lenses (CLs). While the proteins in the tears have been extensively studied with various proteomic techniques, adhered proteins to CLs are less studied. In this pilot study, we have separated proteins with 2D gel electrophoresis prior to the conventional mass spectrometry (MS) in order to analyse the deposited proteins on hydrogel CLs from myopic patients. pHEMA and PVA hydrogel CLs worn by 3 patients for different time lengths were analysed. After wear, the CLs were frozen at -20°C. Proteins were extracted in lysis buffer, separated on 12% polyacrylamide gels and silver-stained. Protein spots were excised and identified with liquid chromatography - tandem MS. Deposited proteins were extracted with a yield of 26-66 μg and separated by 2D gel electrophoresis. The silver-stained gels showed similar protein patterns independent of the patient, hydrogel type and wear time. Seventy-two spots were analysed with MS, representing at least 12 different tear film proteins or protein fragments. Deposited tear film proteins from a single set of CLs worn for 1 day can successfully be analysed first with 2D gel electrophoresis and subsequently with MS, thus making examination of individual patients possible. The protein composition appeared homogeneous between the test persons which is a necessity for additional comparison analysis. The molecular masses of the identified proteins indicate that protein degradation occurs only as a minor event. Myopic patients were investigated in this pilot study, but the combined techniques can easily be applied to other eye diseases. © 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  5. A gel electrophoresis loading system to prevent laboratory contamination by amplification products.

    PubMed

    Adey, Nils B; Emery, Dale B; Bosh, Derek D; Parry, Robert J

    2014-10-01

    Pipet tip loading of polymerase chain reaction (PCR) and other amplification products into an electrophoresis gel represents a potential source of laboratory contamination. We have developed a prototype of the gel contamination control system (GelCCS) that enables gel loading by bottom puncture of PCR tubes. Puncture occurs within a sealed gel casing, preventing contamination of the surrounding environment. The system was designed for inexpensive manufacture so that after the results are visualized, the gel casing and PCR tubes are discarded intact with the amplification products sealed inside. We demonstrate that gel loading is reliable and that the resulting bands are equivalent in appearance to manually loaded gels.

  6. Two-dimensional fluorescence difference gel electrophoresis analysis of Listeria monocytogenes submitted to a redox shock.

    PubMed

    Ignatova, Maria; Guével, Blandine; Com, Emmanuelle; Haddad, Nabila; Rossero, Albert; Bogard, Philippe; Prévost, Hervé; Guillou, Sandrine

    2013-02-21

    The influence of redox alteration on the growth and proteomic pattern of Listeria monocytogenes was investigated. A redox shock was induced in cultures by addition of 3mM ferricyanide (FeCN) and 6mM dithiothreitol (DTT) to increase or to decrease respectively the redox potential naturally occurring at the beginning of growth. In both conditions, the reducing and oxidizing redox shock had a strong influence, decreasing the maximum growth rate by half compared to a control culture. The proteomic analysis of L. monocytogenes performed by two-dimensional difference gel electrophoresis (2D-DIGE) exhibited twenty-three proteins differentially expressed (P<0.05), among these, many were oxidoreductases, and proteins involved in cellular metabolism (glycolysis, protein synthesis), detoxification (kat) or adhesion (Lmo1634).

  7. Proteomic profiling of the mesenteric lymph after hemorrhagic shock: Differential gel electrophoresis and mass spectrometry analysis

    PubMed Central

    2011-01-01

    Experiments show that upon traumatic injury the composition of mesenteric lymph changes such that it initiates an immune response that can ultimately result in multiple organ dysfunction syndrome (MODS). To identify candidate protein mediators of this process we carried out a quantitative proteomic study on mesenteric lymph from a well characterized rat shock model. We analyzed three animals using analytical 2D differential gel electrophoresis. Intra-animal variation for the majority of protein spots was minor. Functional clustering of proteins revealed changes arising from several global classes that give novel insight into fundamental mechanisms of MODS. Mass spectrometry based proteomic analysis of proteins in mesenteric lymph can effectively be used to identify candidate mediators and loss of protective agents in shock models. PMID:21906351

  8. Horizontal comparative fluorescence two-dimensional gel electrophoresis for improved spot coordinate detection.

    PubMed

    Hanneken, Marina; König, Simone

    2014-04-01

    Vertical comparative 2D fluorescence gel electrophoresis (CoFGE) has recently been shown to increase the reproducibility of coordinate assignment for protein spots, in particular in singular experiments, which cannot be investigated using DIGE. The method applies a standardized marker grid formed by a set of purified proteins to the sample proteome in a conglomerate of 1DE, 2DE, and DIGE. Here, improvements are demonstrated by transferring CoFGE to horizontal 2DE. These include the elimination of the protein modification by residual acrylamide monomer unavoidable in vertical CoFGE, reduced buffer volumes, and highly efficient laboratory procedures. Spot patterns are well defined and can be easily analyzed using commercially available warping algorithms. With horizontal CoFGE also a correction for changes in pI was introduced using a third fluorescent dye. Horizontal CoFGE holds high promises in comparative proteomics.

  9. A simple system for staining protein and nucleic acid electrophoresis gels.

    PubMed

    Raymer, Dorian M; Smith, Douglas E

    2007-03-01

    Researchers in molecular biology spend a significant amount of time tending to the staining and destaining of electrophoresis gels. Here we describe a simple system, costing approximately $100 and taking approximately 1 h to assemble, that automates standard nucleic acid and protein gel staining protocols. Staining is done in a tray or, with DNA gels, in the electrophoresis chamber itself following automatic detection of the voltage drop. Miniature pumps controlled by a microcontroller chip exchange the necessary solutions at programmed time intervals. We demonstrate efficient and highly reproducible ethidium bromide and methylene blue staining of DNA in agarose gels and Coomassie blue and silver staining of proteins in polyacrylamide gels.

  10. Characterization of wine rennet and its kinetics by gel electrophoresis.

    PubMed

    Jiang, T M; Chen, L J

    2010-03-01

    The rennet of glutinous rice wine (wine rennet) is an exclusive clotting agent for Chinese Royal cheese production. Some characterizations are reported herein in an attempt to provide evidence about the use of the protease as either a rennet substitute or an accelerator in cheese making and ripening. The results showed that wine rennet was a monomeric and unglycosylated protease. The N-sequencing indicated a high degree of similarity to other fungal rennets. The cleavage sites of wine rennet on oxidized insulin B chain identified by HPLC-mass spectrometry included Gln(4)-His(5), Ala(14)-Leu(15), Leu(15)-Tyr(16), Tyr(16)-Leu(17), and Phe(24)-Phe(25) at pH 6.5, which were similar to those observed for Mucor rennet, but different from calf chymosin except for Leu(15)-Tyr(16). A comparison study of the kinetic properties of wine rennet on bovine caseins with that of rennets from calf and Mucor miehei by gel electrophoresis showed that these rennets had similar coagulation efficiency but different reaction rates. Wine rennet exhibited a higher degree of degradation than the calf and Mucor enzymes at pH 6.5 and 40 degrees C. Therefore, wine rennet would be an adjunct for calf rennet or an accelerator in cheese making.

  11. Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

    PubMed Central

    Hillebrand, G G; McCluskey, A H; Abbott, K A; Revich, G G; Beattie, K L

    1984-01-01

    A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme. Images PMID:6326053

  12. Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis.

    PubMed

    Kosar-Hashemi, Behjat; Irwin, Jennifer A; Higgins, Jody; Rahman, Sadequr; Morell, Matthew K

    2006-05-01

    A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.

  13. Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: II. Experimental results.

    PubMed

    Shen, Hong; Li, Xiang; Bieberich, Charles; Frey, Douglas D

    2009-02-06

    Part I of this study investigated the theory and basic characteristics of "serial displacement chromatofocusing" (SDC). In Part II of this study, SDC is applied to two prototype applications which have potential uses in proteomics and related areas involving the analysis of complex analyte mixtures. In the first application, SDC was used as a prefractionation method prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate a human prostate cancer cell lysate. It was observed that the resolution achieved in narrow-pI-range 2D-PAGE was improved when using SDC prefractionation, so that SDC may be useful as a low-cost, high-speed, and highly scalable alternative to electrophoretic prefractionation methods for 2D-PAGE. The second application involves the use of SDC as the first dimension, and reversed-phase chromatography as the second dimension, to produce a novel, fully automated, two-dimensional high-performance liquid chromatography technique. The method was shown to have performance advantages over one-dimensional reversed-phase chromatography for peptide separations.

  14. Resolution of high molecular weight proteins in dependence on electric field strength in polyacrylamide gel electrophoresis.

    PubMed

    Starita-Geribaldi, M; Houri, A

    1997-01-01

    Resolution of high molecular weight proteins, in the upper region of polyacrylamide gels, was studied in relation to the type of electric field. Separations by constant field gel electrophoresis (CFGE) were compared to those in pulsed oscillatory high-performance electrophoresis (POPE), a novel technique which allows electrophoresis at high field strengths owing to a novel local field distribution. This distribution contributes to structural and mechanical stability of the gel with resultant well-reproducible separation, enhanced resolution, and higher absolute mobility of proteins in POPE.

  15. Analysis of variations in band positions for normalization in across-gel denaturing gradient gel electrophoresis.

    PubMed

    Matsushita, Yuko; Yamamura, Kohji; Morimoto, Sho; Bao, Zhihua; Kurose, Daisuke; Sato, Ikuo; Yoshida, Shigenobu; Tsushima, Seiya

    2015-05-01

    Variation in band position between gels is a well-known problem in denaturing gradient gel electrophoresis (DGGE). However, few reports have evaluated the degree of variation in detail. In this study, we investigated the variation in band positions of DNA samples extracted from soil, normalized using reference positions within marker lanes for DGGE in three organismal (bacterial, fungal, and nematode) conditions. For sample lanes, marker DNA (as a control) and sample DNA were used. The test for normality of distribution showed that the position data of a large percentage of bands were normally distributed but not for certain bands. For the normally-distributed data, their variations [standard deviation of marker bands (SDM) and standard deviation of sample bands (SDS), respectively] were assessed. For all organismal conditions, the degree of within-gel variation were similar between SDMs and SDSs, while between-gel variations in SDSs were larger than those in SDMs. Due to the large effect of between-gel variations, the total variations in SDSs were more varied between sample bands, and the mean variations of all sample bands were higher than those in the markers. We found that the total variation in the fungal and nematode SDSs decreased when the intervals between marker bands were narrowed, suggesting that band interval is important for reducing total variation in normalized band positions. For the non-normally distributed data, the distribution was examined in detail. This study provided detailed information on the variation of band positions, which could help to optimize markers for reducing band position variation, and could aid in the accurate identification of bands in across-gel DGGE analyses.

  16. Congruence between starch gel and polyacrylamide gel electrophoresis in detecting allozyme variation in pulmonate land slugs.

    PubMed

    Geenen, Sofie; Jordaens, Kurt; Castilho, Rita; Backeljau, Thierry

    2003-02-01

    The predominantly selfing slug species Arion (Carinarion) fasciatus, A. (C.) silvaticus and A. (C.) circumscriptus are native in Europe and have been introduced into North America, where each species consists of a single, homozygous multilocus genotype (strain), as defined by starch gel electrophoresis (SGE) of allozymes. In Europe, the "one strain per species" hypothesis does not hold since polyacrylamide gel electrophoresis (PAGE) of allozymes uncovered 46 strains divided over the three species. However, electrophoretic techniques may differ in their ability to detect allozyme variation. Therefore, several Carinarion populations from both continents were screened by applying the two techniques simultaneously on the same individual slugs and enzyme loci. SGE and PAGE yielded exactly the same results, so that the different degree of variation in North American and European populations cannot be attributed to differences in resolving power between SGE and PAGE. We found four A. (C.) silvaticus strains in North America indicating that in this region the "one strain per species" hypothesis also cannot be maintained. Hence, the discrepancies between previous electrophoretic studies on Carinarion are most likely due to sampling artefacts and possible founder effects.

  17. Retardation of serum albumin migration in native polyacrylamide gel electrophoresis by the incorporation of blue dextran in the gel matrix.

    PubMed

    Pemberton, A D; John, H A

    1993-03-01

    Blue dextran was incorporated into the stacking layer of one-dimensional polyacrylamide gels to prevent the migration of albumin into the resolving gel during the electrophoresis of native plasma samples. Human and horse plasma proteins normally masked by albumin were revealed by this method.

  18. Binding of lithium dodecyl sulfate to polyacrylamide gel at 4 degrees C perturbs electrophoresis of proteins.

    PubMed

    Kubo, K; Takagi, T

    1986-07-01

    Although polyacrylamide gel has no affinity to lithium dodecyl sulfate (LDS) at 25 degrees C, the gel maximally binds 17 mg of LDS per gram dry weight at 4 degrees C. When polyacrylamide gel electrophoresis is carried out at 4 degrees C in the presence of LDS instead of sodium dodecyl sulfate (SDS) using a continuous buffer system, migration of proteins with lower molecular weight is accelerated as a result of the deficiency of LDS in the frontal region of the gel. When the gel is saturated with LDS, electrophoresis in the presence of LDS at 4 degrees C shows a resolution higher than that of SDS-polyacrylamide gel electrophoresis at 25 degrees C.

  19. Analysis of tissue proteomes of the Gulf killifish, Fundulus grandis, by 2D electrophoresis and MALDI-TOF/TOF mass spectrometry.

    PubMed

    Abbaraju, Naga V; Boutaghou, Mohamed Nazim; Townley, Ian K; Zhang, Qiang; Wang, Guangdi; Cole, Richard B; Rees, Bernard B

    2012-11-01

    The Gulf killifish, Fundulus grandis, is a small teleost fish that inhabits marshes of the Gulf of Mexico and demonstrates high tolerance of environmental variation, making it an excellent subject for the study of physiological and molecular adaptations to environmental stress. In the present study, two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry were used to resolve and identify proteins from five tissues: skeletal muscle, liver, brain, heart, and gill. Of 864 protein features excised from 2D gels, 424 proteins were identified, corresponding to a 49% identification rate. For any given tissue, several protein features were identified as the same protein, resulting in a total of 254 nonredundant proteins. These nonredundant proteins were categorized into a total of 11 molecular functions, including catalytic activity, structural molecule, binding, and transport. In all tissues, catalytic activity and binding were the most highly represented molecular functions. Comparing across the tissues, proteome coverage was lowest in skeletal muscle, due to a combination of a low number of gel spots excised for analysis and a high redundancy of identifications among these spots. Nevertheless, the identification of a substantial number of proteins with high statistical confidence from other tissues suggests that F. grandis may serve as a model fish for future studies of environmental proteomics and ultimately help to elucidate proteomic responses of fish and other vertebrates to environmental stress.

  20. Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels.

    PubMed

    Wrigley, C W; Margolis, J

    1992-01-01

    Precast gradient gels of short migration length (25 mm) have been developed to provide rapid electrophoretic separation without loss of resolution. These Micrograd gels have been prepared in gel ranges (conventional and unique) to match pore-gradient electrophoresis conditions to proteins/peptides ranging in size from several hundreds to millions. The Hylinx Micrograd gel combines an extreme gel range (6 to 48% polyacrylamide) with a novel crosslinker to provide sieving of polypeptides, and pore-limit electrophoresis of the smallest proteins (e.g. insulin monomer). All gel ranges (such as 3 to 30%) provide zone sharpening in routine analysis of conventional protein mixtures (e.g. serum) within 10 min electrophoresis at 200 to 300 volts. The gels are thin (1 mm) and thus stain quickly, but the gel cassette is of conventional overall width (83 mm), thus fitting many apparatus designs and accommodating 12 samples. The gels are finding valuable use in screening applications, requiring the electrophoretic analysis of many samples, and in cases where a rapid answer is needed, such as monitoring protein purification. The gels have proved particularly useful, in-house, for the latter application in developing Gradipore's new large-scale preparative electrophoresis system, the Gradiflow.

  1. Nanoparticle gel electrophoresis: soft spheres in polyelectrolyte hydrogels under the Debye-Hückel approximation.

    PubMed

    Li, Fei; Allison, Stuart A; Hill, Reghan J

    2014-06-01

    A mathematical model for electrophoresis of polyelectrolyte coated nanoparticles (soft spheres) in polyelectrolyte hydrogels is proposed, and evaluated by comparison to literature models for bare-sphere gel electrophoresis and free-solution electrophoresis. The utilities of approximations based on the bare-particle electrophoretic mobility, free-solution mobility, and electroosmotic flow in hydrogels are explored. Noteworthy are the influences of the particle-core dielectric constant and overlap of the polyelectrolyte shell. The present theory, which neglects ion-concentration and charge-density perturbations, indicates that the gel electrophoretic mobilities of metallic-core nanoparticles in polyelectrolyte gels can be qualitatively different than for their non-metallic counterparts. These insights will be beneficial for interpreting nanoparticle gel-electrophoresis data, optimizing electrophoretic separations, and engineering nanoparticles for technological applications. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. The latest advancements in proteomic two-dimensional gel electrophoresis analysis applied to biological samples.

    PubMed

    Santucci, Laura; Bruschi, Maurizio; Ghiggeri, Gian Marco; Candiano, Giovanni

    2015-01-01

    Two-dimensional gel electrophoresis (2DE) is one of the fundamental approaches in proteomics for the separation and visualization of complex protein mixtures. Proteins can be analyzed by 2DE using isoelectric focusing (IEF) in the first dimension, combined to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, gel staining (silver and Coomassie), image analysis, and 2DE gel database. High-resolution 2DE can resolve up to 5,000 different proteins simultaneously (∼2,000 proteins routinely), and detect and quantify <1 ng of protein per spot. Here, we describe the latest developments for a more complete analysis of biological fluids.

  3. Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis.

    PubMed

    Pisani, Francesca; Livermore, Thomas; Rose, Giuseppina; Chubb, Jonathan Robert; Gaspari, Marco; Saiardi, Adolfo

    2014-01-01

    The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba

  4. Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis

    PubMed Central

    Pisani, Francesca; Livermore, Thomas; Rose, Giuseppina; Chubb, Jonathan Robert; Gaspari, Marco; Saiardi, Adolfo

    2014-01-01

    The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP6 or Phytic acid) and its derivative inositol pyrophosphates, IP7 and IP8. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP9 in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP5) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP8 was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed

  5. Insight of Saffron Proteome by Gel-Electrophoresis.

    PubMed

    Paredi, Gianluca; Raboni, Samanta; Marchesani, Francesco; Ordoudi, Stella A; Tsimidou, Maria Z; Mozzarelli, Andrea

    2016-01-29

    Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds.

  6. Characterization of lactosylated proteins of infant formula powders using two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry.

    PubMed

    Marvin, Laure F; Parisod, Véronique; Fay, Laurent B; Guy, Philippe A

    2002-08-01

    Infant formula powders were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to assess the whey proteins quality, which may be altered by the heat treatment used during the processing conditions. Lactosylation was found to be the major chemical modification occurring in whey proteins. In parallel, a two-dimensional (2-D) gel electrophoresis was performed on the milk sample and the entire protein patterns were analyzed by nano-ESI-MS after cutting the different gel spots and in-gel trypsin digestion. A highly selective and specific tandem MS technique has been developed to characterize and localize up to ten lactosylation sites in beta-lactoglobulin (beta-Lg) and alpha(S2)-casein. alpha-Lactalbumin (alpha-La), with five lactosylated peptides, was found to be an interesting protein marker in the milk powder sample to detect chemical modification induced by the processing/storage conditions.

  7. Rheological and mechanical behavior of polyacrylamide hydrogels chemically crosslinked with allyl agarose for two-dimensional gel electrophoresis.

    PubMed

    Suriano, R; Griffini, G; Chiari, M; Levi, M; Turri, S

    2014-02-01

    Two-dimensional (2-D) gel electrophoresis currently represents one of the most standard techniques for protein separation. In addition to the most commonly employed polyacrylamide crosslinked hydrogels, acrylamide-agarose copolymers have been proposed as promising systems for separation matrices in 2-D electrophoresis, because of the good resolution of both high and low molecular mass proteins made possible by careful control and optimization of the hydrogel pore structure. As a matter of fact, a thorough understanding of the nature of the hydrogel pore structure as well as of the parameters by which it is influenced is crucial for the design of hydrogel systems with optimal sieving properties. In this work, a series of acrylamide-based hydrogels covalently crosslinked with different concentrations of allyl agarose (0.2-1%) is prepared and characterized by creep-recovery measurements, dynamic rheology and tensile tests, in the attempt to gain a clearer understanding of structure-property relationships in crosslinked polyacrylamide-based hydrogels. The rheological and mechanical properties of crosslinked acrylamide-agarose hydrogels are found to be greatly affected by crosslinker concentration. Dynamic rheological tests show that hydrogels with a percentage of allyl agarose between 0.2% and 0.6% have a low density of elastically effective crosslinks, explaining the good separation of high molecular mass proteins in 2-D gel electrophoresis. Over the same range of crosslinker concentration, creep-recovery measurements reveal the presence of non-permanent crosslinks in the hydrogel network that justifies the good resolution of low molecular mass proteins as well. In tensile tests, the hydrogel crosslinked with 0.4% of allyl agarose exhibits the best results in terms of mechanical strength and toughness. Our results show how the control of the viscoelastic and the mechanical properties of these materials allow the design of mechanically stable hydrogels with improved

  8. [THE USE OF THE COMMERCIAL APPARATUS DUAL GEL MODULE FOR THE TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS].

    PubMed

    Evteeva, I N; Starkova, T Yu; Artemov, A V; Barlev, N A

    2015-01-01

    Two-dimensional gel electrophoresis, continues to be one of the fundamental methods to study the biological protein diversity. This method described by O'Farrell in 1975 includes two following steps: isoelectric focusing in the first dimension and polyacrylamide gel electrophoretic fractionation of proteins according to their molecular weight in the second dimension. In this manuscript we described several technical parameters of the commercial apparatus Dual Gel Module for the gel electrophoresis by means of which it is possible to accomplish the electrophoretic protein fractionation in both dimensions. The distribution of the highly purified commercial proteins used as molecular standards in the detection system of the apparatus Dual Gel Module was identical to the commercial strips of the device GE Healthcare, USA.

  9. Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

    PubMed Central

    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 (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of 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 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 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. PMID:22222979

  10. Proteomic Profile in Glomeruli of Type-2 Diabetic KKAy Mice using 2-Dimensional Differential Gel Electrophoresis

    PubMed Central

    Liu, Xiaodan; Yang, Gang; Fan, Qiuling; Wang, Lining

    2014-01-01

    Background Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. To search for glomerular proteins associated with early-stage DN, glomeruli of spontaneous type 2 diabetic KKAy mice were analyzed by 2-dimensional differential gel electrophoresis (2D-DIGE). Material/Methods Glomeruli of 20-week spontaneous type 2 diabetic KKAy mice and age-matched C57BL/6 mice were isolated by kidney perfusion with magnetic beads. Proteomic profiles of glomeruli were investigated by using 2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Western blot analysis was used to confirm the results of proteomics. Immunohistochemical and semi-quantitative analysis were used to confirm the differential expression of prohibitin and annexin A2 in glomeruli. Results We identified 19 differentially expressed proteins – 17 proteins were significantly up-regulated and 2 proteins were significantly down-regulated in glomeruli of diabetic KKAy mice. Among them, prohibitin and annexin A2 were up-regulated and Western blot analysis validated the same result in proteomics. Immunohistochemical analysis also revealed up-regulation of prohibitin and annexin A2 in glomeruli of KKAy mice. Conclusions Our findings suggest that prohibitin and annexin A2 may be associated with early-stage DN. Further functional research might help to reveal the pathogenesis of DN. PMID:25515740

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

    PubMed

    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.

  12. A Wavelet Relational Fuzzy C-Means Algorithm for 2D Gel Image Segmentation

    PubMed Central

    Rashwan, Shaheera; Faheem, Mohamed Talaat; Sarhan, Amany; Youssef, Bayumy A. B.

    2013-01-01

    One of the most famous algorithms that appeared in the area of image segmentation is the Fuzzy C-Means (FCM) algorithm. This algorithm has been used in many applications such as data analysis, pattern recognition, and image segmentation. It has the advantages of producing high quality segmentation compared to the other available algorithms. Many modifications have been made to the algorithm to improve its segmentation quality. The proposed segmentation algorithm in this paper is based on the Fuzzy C-Means algorithm adding the relational fuzzy notion and the wavelet transform to it so as to enhance its performance especially in the area of 2D gel images. Both proposed modifications aim to minimize the oversegmentation error incurred by previous algorithms. The experimental results of comparing both the Fuzzy C-Means (FCM) and the Wavelet Fuzzy C-Means (WFCM) to the proposed algorithm on real 2D gel images acquired from human leukemias, HL-60 cell lines, and fetal alcohol syndrome (FAS) demonstrate the improvement achieved by the proposed algorithm in overcoming the segmentation error. In addition, we investigate the effect of denoising on the three algorithms. This investigation proves that denoising the 2D gel image before segmentation can improve (in most of the cases) the quality of the segmentation. PMID:24174990

  13. A wavelet relational fuzzy C-means algorithm for 2D gel image segmentation.

    PubMed

    Rashwan, Shaheera; Faheem, Mohamed Talaat; Sarhan, Amany; Youssef, Bayumy A B

    2013-01-01

    One of the most famous algorithms that appeared in the area of image segmentation is the Fuzzy C-Means (FCM) algorithm. This algorithm has been used in many applications such as data analysis, pattern recognition, and image segmentation. It has the advantages of producing high quality segmentation compared to the other available algorithms. Many modifications have been made to the algorithm to improve its segmentation quality. The proposed segmentation algorithm in this paper is based on the Fuzzy C-Means algorithm adding the relational fuzzy notion and the wavelet transform to it so as to enhance its performance especially in the area of 2D gel images. Both proposed modifications aim to minimize the oversegmentation error incurred by previous algorithms. The experimental results of comparing both the Fuzzy C-Means (FCM) and the Wavelet Fuzzy C-Means (WFCM) to the proposed algorithm on real 2D gel images acquired from human leukemias, HL-60 cell lines, and fetal alcohol syndrome (FAS) demonstrate the improvement achieved by the proposed algorithm in overcoming the segmentation error. In addition, we investigate the effect of denoising on the three algorithms. This investigation proves that denoising the 2D gel image before segmentation can improve (in most of the cases) the quality of the segmentation.

  14. High performance CCD camera system for digitalisation of 2D DIGE gels.

    PubMed

    Strijkstra, Annemieke; Trautwein, Kathleen; Roesler, Stefan; Feenders, Christoph; Danzer, Daniel; Riemenschneider, Udo; Blasius, Bernd; Rabus, Ralf

    2016-07-01

    An essential step in 2D DIGE-based analysis of differential proteome profiles is the accurate and sensitive digitalisation of 2D DIGE gels. The performance progress of commercially available charge-coupled device (CCD) camera-based systems combined with light emitting diodes (LED) opens up a new possibility for this type of digitalisation. Here, we assessed the performance of a CCD camera system (Intas Advanced 2D Imager) as alternative to a traditionally employed, high-end laser scanner system (Typhoon 9400) for digitalisation of differential protein profiles from three different environmental bacteria. Overall, the performance of the CCD camera system was comparable to the laser scanner, as evident from very similar protein abundance changes (irrespective of spot position and volume), as well as from linear range and limit of detection. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. High performance CCD camera system for digitalisation of 2D DIGE gels

    PubMed Central

    Strijkstra, Annemieke; Trautwein, Kathleen; Roesler, Stefan; Feenders, Christoph; Danzer, Daniel; Riemenschneider, Udo; Blasius, Bernd

    2016-01-01

    An essential step in 2D DIGE‐based analysis of differential proteome profiles is the accurate and sensitive digitalisation of 2D DIGE gels. The performance progress of commercially available charge‐coupled device (CCD) camera‐based systems combined with light emitting diodes (LED) opens up a new possibility for this type of digitalisation. Here, we assessed the performance of a CCD camera system (Intas Advanced 2D Imager) as alternative to a traditionally employed, high‐end laser scanner system (Typhoon 9400) for digitalisation of differential protein profiles from three different environmental bacteria. Overall, the performance of the CCD camera system was comparable to the laser scanner, as evident from very similar protein abundance changes (irrespective of spot position and volume), as well as from linear range and limit of detection. PMID:27252121

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

    PubMed

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

    2006-03-20

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

  17. Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

    SciTech Connect

    Kovacs, E.; Langemann, H. )

    1990-11-01

    Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of (3H)thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.

  18. Analyzing RNA and DNA folding using temperature gradient gel electrophoresis (TGGE) with application to in vitro selections.

    PubMed

    Chadalavada, Durga M; Bevilacqua, Philip C

    2009-01-01

    Gel electrophoresis is a ubiquitous separation technique in nucleic acid biochemistry. Denaturing gel electrophoresis separates nucleic acids on the basis of length, while native gel electrophoresis separates nucleic acids on the basis of both shape and length. Temperature gradient gel electrophoresis (TGGE), in which a temperature gradient is present across the gel, combines the advantages of denaturing and native gel electrophoresis by having native gel-like properties at low temperatures and denaturing gel-like properties at high temperatures. We describe here the techniques of perpendicular and parallel TGGE and some of their applications. Isolation of stable and unstable RNA and DNA sequences from combinatorial libraries is accomplished with TGGE-SELEX, while thermodynamic characterization of an RNA tertiary motif is performed by perpendicular TGGE-melts. Specific examples are chosen from the literature to illustrate the methods. TGGE provides a powerful biophysical approach for analyzing RNA and DNA that complements more traditional methodologies.

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

    PubMed Central

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

    2014-01-01

    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 upon 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. PMID:24637158

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

    PubMed

    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.

  1. Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state

    PubMed Central

    Migliore, Luciana; Rotini, Alice; Randazzo, Davide; Albanese, Nadia N; Giallongo, Agata

    2007-01-01

    Background The endemic seagrass Posidonia oceanica (L.) Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. Results The overall expression pattern from Posidonia rhizome was determined using a preliminary proteomic approach, 437 protein spots were characterized by pI and molecular weight. We found that protein expression differs in samples belonging to sites with high or low phenols: 22 unique protein spots are peculiar of "low phenols" and 27 other spots characterize "high phenols" samples. Conclusion Posidonia showed phenols variations within the meadow, that probably reflect the heterogeneity of environmental pressures. In addition, comparison of the 2-D electrophoresis patterns allowed to highlight qualitative protein expression differences in response to these pressures. These differences may account for changes in metabolic/physiological pathways as adaptation to stress. A combined approach, based on phenols content determination and 2-D electrophoresis protein pattern, seems a promising tool to monitor Posidonia meadows health state. PMID:17663776

  2. Sodium dodecyl sulfate-agarose gel electrophoresis for the detection and isolation of amyloid curli fibers.

    PubMed

    Sitaras, Chris; Naghavi, Mahsa; Herrington, Muriel B

    2011-01-15

    Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)-agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS-agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS-agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS-agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available.

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

    PubMed

    Zakharchenko, Olena; Greenwood, Christina; Alldridge, Louise; Souchelnytskyi, Serhiy

    2011-03-10

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

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

    PubMed

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

    2013-12-19

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

  5. Application of multiplexed cysteine-labeled complex protein sample for 2D electrophoretic gel alignment.

    PubMed

    Haimi, Perttu; Sikorskaite-Gudziuniene, Sidona; Baniulis, Danas

    2015-06-01

    The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost-effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co-run with the sample. Maleimide-conjugated fluorescent dyes Dy-560 and Dy-635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.

  6. A Microcomputer Based Charge Coupled Device (CCD) Imaging System For Clinical 2D Electrophoretic Gel Analysis

    NASA Astrophysics Data System (ADS)

    Craine, Brian L.; Craine, Eric R.; Engel, John R.; Wemple, Neil T.

    1988-06-01

    We have developed a prototype microcomputer based gel reader for the digitization and semi-automatic analysis of two dimensional polyacrylamide electrophoretic gels. The system uses a 2D charge coupled device (CCD) detector and operates as a peripheral to an IBM PC XT/AT type host computer. The system incorporates a comprehensive software package which lends itself to highly structured clinical applications as well as a wide range of image processing functions for use in the research laboratory. The system is undergoing further development in order to arrive at a comprehensive, low cost, early disease screening device. Electrophoretic gel samples of protein components obtained from subject patients are digitized and searched for anomalous features. These features are located using a digital reference library of disease related protein features and a probability of detection of the disease related protein is computed. The system then presents the physician with a report advising him of the results of the analysis. We describe the hardware and software system and present results of a sample gel analysis.

  7. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

    Zhang, Jian-Shi; Giometti, Carol S.; Tollaksen, Sandra L.

    1989-01-01

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

  8. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    SciTech Connect

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

    1989-04-25

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower and of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

  9. Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique.

    PubMed

    Intarapanich, Apichart; Kaewkamnerd, Saowaluck; Shaw, Philip J; Ukosakit, Kittipat; Tragoonrung, Somvong; Tongsima, Sissades

    2015-01-01

    DNA gel electrophoresis is a molecular biology technique for separating different sizes of DNA fragments. Applications of DNA gel electrophoresis include DNA fingerprinting (genetic diagnosis), size estimation of DNA, and DNA separation for Southern blotting. Accurate interpretation of DNA banding patterns from electrophoretic images can be laborious and error prone when a large number of bands are interrogated manually. Although many bio-imaging techniques have been proposed, none of them can fully automate the typing of DNA owing to the complexities of migration patterns typically obtained. We developed an image-processing tool that automatically calls genotypes from DNA gel electrophoresis images. The image processing workflow comprises three main steps: 1) lane segmentation, 2) extraction of DNA bands and 3) band genotyping classification. The tool was originally intended to facilitate large-scale genotyping analysis of sugarcane cultivars. We tested the proposed tool on 10 gel images (433 cultivars) obtained from polyacrylamide gel electrophoresis (PAGE) of PCR amplicons for detecting intron length polymorphisms (ILP) on one locus of the sugarcanes. These gel images demonstrated many challenges in automated lane/band segmentation in image processing including lane distortion, band deformity, high degree of noise in the background, and bands that are very close together (doublets). Using the proposed bio-imaging workflow, lanes and DNA bands contained within are properly segmented, even for adjacent bands with aberrant migration that cannot be separated by conventional techniques. The software, called GELect, automatically performs genotype calling on each lane by comparing with an all-banding reference, which was created by clustering the existing bands into the non-redundant set of reference bands. The automated genotype calling results were verified by independent manual typing by molecular biologists. This work presents an automated genotyping tool from DNA

  10. Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique

    PubMed Central

    2015-01-01

    Background DNA gel electrophoresis is a molecular biology technique for separating different sizes of DNA fragments. Applications of DNA gel electrophoresis include DNA fingerprinting (genetic diagnosis), size estimation of DNA, and DNA separation for Southern blotting. Accurate interpretation of DNA banding patterns from electrophoretic images can be laborious and error prone when a large number of bands are interrogated manually. Although many bio-imaging techniques have been proposed, none of them can fully automate the typing of DNA owing to the complexities of migration patterns typically obtained. Results We developed an image-processing tool that automatically calls genotypes from DNA gel electrophoresis images. The image processing workflow comprises three main steps: 1) lane segmentation, 2) extraction of DNA bands and 3) band genotyping classification. The tool was originally intended to facilitate large-scale genotyping analysis of sugarcane cultivars. We tested the proposed tool on 10 gel images (433 cultivars) obtained from polyacrylamide gel electrophoresis (PAGE) of PCR amplicons for detecting intron length polymorphisms (ILP) on one locus of the sugarcanes. These gel images demonstrated many challenges in automated lane/band segmentation in image processing including lane distortion, band deformity, high degree of noise in the background, and bands that are very close together (doublets). Using the proposed bio-imaging workflow, lanes and DNA bands contained within are properly segmented, even for adjacent bands with aberrant migration that cannot be separated by conventional techniques. The software, called GELect, automatically performs genotype calling on each lane by comparing with an all-banding reference, which was created by clustering the existing bands into the non-redundant set of reference bands. The automated genotype calling results were verified by independent manual typing by molecular biologists. Conclusions This work presents an

  11. Two-dimensional difference gel electrophoresis (DiGE) analysis of plasmas from dengue fever patients.

    PubMed

    Albuquerque, Lidiane M; Trugilho, Monique R O; Chapeaurouge, Alex; Jurgilas, Patrícia B; Bozza, Patrícia T; Bozza, Fernando A; Perales, Jonas; Neves-Ferreira, Ana G C

    2009-12-01

    Dengue fever is the world's most important arthropod-born viral disease affecting humans. To contribute to a better understanding of its pathogenesis, this study aims to identify proteins differentially expressed in plasmas from severe dengue fever patients relative to healthy donors. The use of 2-D Fluorescence Difference Gel Electrophoresis to analyze plasmas depleted of six high-abundance proteins (albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin) allowed for the detection of 73 differentially expressed protein spots (n = 13, p < 0.01), of which 37 could be identified by mass spectrometry. These 37 spots comprised a total of 14 proteins, as follows: 7 had increased expression in plasmas from dengue fever patients (C1 inhibitor, alpha1-antichymotrypsin, vitamin D-binding protein, fibrinogen gamma-chain, alpha1-acid glycoprotein, apolipoprotein J and complement component C3c), while 7 others had decreased expression in the same samples (alpha-2 macroglobulin, prothrombin, histidine-rich glycoprotein, apolipoproteins A-IV and A-I, transthyretin and complement component C3b). The possible involvement of these proteins in the inflammatory process triggered by dengue virus infection and in the repair mechanisms of vascular damage occurring in this pathology is discussed in this study.

  12. 2-D gel-based proteomic approaches to antibiotic drug discovery.

    PubMed

    Raatschen, Nadja; Bandow, Julia Elisabeth

    2012-08-01

    The global analysis of changes in the protein composition of bacterial cells in response to treatment with antibiotic agents grants insights into the physiological response of cells to inhibition of vital cellular functions. This unit gives an overview of how global proteomic studies can impact antibacterial drug discovery by identifying or validating compound mechanism of action and by increasing the confidence in the value of genes with unknown function as potential new targets. It describes the design and function of a reference compendium of proteomic responses to inhibition of vital cellular functions through antibacterial agents or genetic down-regulation of potential target genes. An overview of the workflow for two-dimensional gel electrophoresis-based experiments is also presented. © 2012 by John Wiley & Sons, Inc.

  13. Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels.

    PubMed

    Cole, Kenneth D; Gaigalas, Adolfas; Akerman, Björn

    2006-11-01

    The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.

  14. Analysis of supercoiled DNA by agarose gel electrophoresis using low-conducting sodium threonine medium.

    PubMed

    Ishido, Tomomi; Ishikawa, Mitsuru; Hirano, Ken

    2010-05-01

    We describe a new low-ionic-strength sodium threonine (STh) medium with the advantage of avoiding relative DNA band migration changes following electrophoresis of supercoiled DNA in agarose gel when substituted for the standard conductive medium of TBE (Tris-boric acid-ethylenediaminetetraacetic acid [EDTA]) or TAE (Tris-acetic acid-EDTA) or the low-ionic-strength sodium boric acid medium. Low-ionic-strength STh medium provided better resolution, less heat generation, and prevention of relative migration order changes among linear, covalently closed circular-, and open circular-formed DNA in the range of 2-10 kilobase pairs in 1% agarose gel electrophoresis.

  15. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

    Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.

    1987-09-04

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

  16. Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

    PubMed

    Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

    2013-09-15

    Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes.

  17. Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Peng, Fang; Li, Jianglin; Guo, Tianyao; Yang, Haiyan; Li, Maoyu; Sang, Shushan; Li, Xuejun; Desiderio, Dominic M.; Zhan, Xianquan

    2015-12-01

    Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-β-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of β-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the

  18. Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry.

    PubMed

    Peng, Fang; Li, Jianglin; Guo, Tianyao; Yang, Haiyan; Li, Maoyu; Sang, Shushan; Li, Xuejun; Desiderio, Dominic M; Zhan, Xianquan

    2015-12-01

    Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-β-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of β-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the

  19. Resolving Acetylated and Phosphorylated Proteins by Neutral Urea Triton-Polyacrylamide Gel Electrophoresis, NUT-PAGE

    PubMed Central

    Buehl, Christopher J.; Deng, Xiexiong; Liu, Mengyu; Hovde, Stacy; Xu, Xinjing; Kuo, Min-Hao

    2014-01-01

    Protein acetylation and phosphorylation can be key modifications that regulate both normal and pathological protein functions. Current gel systems used to analyze modified proteins require either expensive reagents or time–consuming second dimension electrophoresis. In this manuscript, we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. This neutral pH urea Triton-polyacrylamide gel electrophoresis system, or NUT-PAGE, separates proteins based on their charge at pH 7 and generates discrete bands from each acetylated and phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents, and requires only a single dimension of electrophoresis. We are able to demonstrate the effectiveness of this system by analyzing phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative to resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge. Method Summary Here we present a single-dimension neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system affording high-resolution separation of acetylated and phosphorylated proteins. PMID:25109292

  20. Capillary electrophoresis of peptides and proteins with plug of Pluronic gel.

    PubMed

    Sedlakova, P; Svobodova, J; Miksik, I

    2006-07-24

    Electromigration capillary methods are promising techniques in proteomics and they are still under research. We used a partial filling approach, i.e. a combination of gel and non-gel separation mechanisms in a single dimension. We tried using an interesting gel, Pluronic F 127, which can be considered as a surfactant capable of self-association both with isotropic and anisotropic gels. The Pluronic was inserted inside the capillary as a plug at the start of the capillary, and it provided separation at the first time. Separation by this gel was achieved according to molecular weight and/or hydrophobicity. The applicability of this method was demonstrated in the separation of real samples-peptides arising from collagen after CNBr or collagenase cleavage and albumin after trypsin cleavage (peptide mapping). Some peptides and proteins were selectively retained by the Pluronic gel. These interactions with the gel did not depended on their molecular weight alone, but they probably depend on a combination of both principles. It was confirmed that capillary electrophoresis with Pluronic plug can give us another new separation option, complementary to free solution capillary electrophoresis. The CE method presented here, consisting of a partial filling approach with combine gel and non-gel separation mechanisms seemed to be a promising method for the separation of complex mixtures of peptides.

  1. Effect of ultrasound on the separation of DNA fragments in agarose gel electrophoresis

    SciTech Connect

    Ma, Yinfa; Yeung, E.S. )

    1990-06-01

    Since its first use in 1966 interest in and the applications of electrophoresis of DNA fragments in agarose gel have grown rapidly. Nowadays, agarose gel electrophoresis has become a standard technique with high resolving power for the analysis of DNA structure, for example for the determination of the length of DNA fragments obtained by the action of restriction enzymes. The electrophoretic mobility ({mu}) of DNA fragments is influenced by various parameters-molecular weight, gel concentration, temperature, electric field, and DNA-agarose affinity. A comprehensive study of the influence of these main parameters has been reported. In this paper, the authors investigate a new effect on the electrophoretic mobility of DNA fragments in agarose gels, viz. the influence of ultrasound.

  2. Reovirus-specific polypeptides: analysis using discontinuous gel electrophoresis.

    PubMed Central

    Cross, R K; Fields, B N

    1976-01-01

    The electrophoretic analysis of reovirus-specific polypeptides in infected cells using a discontinuous gel system has allowed the resolution of additional viral-specific polypeptides, including one large-sized gamma3 and two (or possibly three) medium-sized (mu3, mu4, mu5(?)) species. The proteins designated mu0, sigma1, and sigma2 based on electrophoretic mobility in gel systems containing phosphate-urea correspond to mu4, sigma2, and sigma1, respectively, when analyzed in systems containing Tris-glycine. It is likely that protein modifications (phosphorylation and glycosylation) are responsible for at least some of these differences. Images PMID:950684

  3. Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques.

    PubMed

    Bocian, Aleksandra; Urbanik, Małgorzata; Hus, Konrad; Łyskowski, Andrzej; Petrilla, Vladimír; Andrejčáková, Zuzana; Petrillová, Monika; Legáth, Jaroslav

    2016-12-13

    Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A₂ and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5'-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor.

  4. Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques

    PubMed Central

    Bocian, Aleksandra; Urbanik, Małgorzata; Hus, Konrad; Łyskowski, Andrzej; Petrilla, Vladimír; Andrejčáková, Zuzana; Petrillová, Monika; Legáth, Jaroslav

    2016-01-01

    Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A2 and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5′-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor. PMID:27983581

  5. Diffusion, Dispersion, and Mobility of Single-stranded DNA in Polyacrylamide Gel Electrophoresis

    NASA Astrophysics Data System (ADS)

    Lo, Roger; Ugaz, Victor

    2004-03-01

    The ability to perform DNA electrophoresis in miniaturized microfluidic systems has the potential to provide a new generation of low-cost high-throughput genomic analysis technology. Further progress toward improving separation performance under these conditions, however, requires a more detailed understanding of diffusion and dispersion phenomena in the gel matrix. Unfortunately, it has thus far proven difficult to obtain extensive measurements of these quantities due in large part to the lack of a convenient experimental platform. In this paper, we demonstrate the use of microfabricated gel electrophoresis devices to measure diffusion, dispersion, and mobility of single-stranded DNA fragments in crosslinked and uncrosslinked polyacrylamide gels. The microdevice format allows a complete set of diffusion and dispersion data to be collected in approximately one hour, as opposed to experiment times lasting several days using conventional sequencing equipment. By comparing runs using identical DNA samples, gel formulations, and operating conditions in both microfabricated electrophoresis devices and an ALF Express automated DNA sequencer, we are able to isolate the key factors governing separation performance in each system. The results of these experiments are then compared with biased reptation theory to extract information about the gel structure and predict achievable resolution. The effects of gel composition and polymerization chemistry are also explored.

  6. Aligning Goals, Assessments, and Activities: An Approach to Teaching PCR and Gel Electrophoresis

    ERIC Educational Resources Information Center

    Phillips, Allison R.; Robertson, Amber L.; Batzli, Janet; Harris, Michelle; Miller, Sarah

    2008-01-01

    Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments…

  7. Beverage-Agarose Gel Electrophoresis: An Inquiry-Based Laboratory Exercise with Virtual Adaptation

    ERIC Educational Resources Information Center

    Cunningham, Steven C.; McNear, Brad; Pearlman, Rebecca S.; Kern, Scott E.

    2006-01-01

    A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As…

  8. Rapid high-resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus.

    PubMed

    Smejkal, Gary B; Shainoff, John R; Kottke-Marchant, Kandice M

    2003-02-01

    Rapid and highly reproducible nonreducing agarose gel electrophoresis (NRAGE) of von Willebrand Factor (vWF) multimers was performed using a thermostated minigel apparatus that monitors and precisely controls internal gel temperature. The substitution of lithium dodecyl sulfate (LiDS) for sodium dodecyl sulfate (SDS) allowed electrophoresis to be performed below the 16 degrees C Krafft point of SDS and facilitated NRAGE of vWF over the entire range of 0-35 degrees C. Internal gel temperature was regulated by a thermocouple probe inserted directly into the gel during electrophoresis which interfaced with a thermopilot that continually measures and adjusts temperature to within +/- 0.5 degrees C. At 10 degrees C operative temperature, NRAGE at 1.5% agarose concentration was completed in 20 min at 250 V. Electrophoresis could be performed in only 10 min at 500 V, but at such high voltages, localized temperature fluctuations as much as 6 degrees C resulted in perturbation of banding patterns in those vicinities. In the optimized method, both high molecular weight multimers and proteolytic fragments of vWF were separable suggesting clinical applicability of this system for the diagnosis of von Willebrand Disease and thrombotic thrombocytopenic purpura.

  9. [The development of an image analysis system of medical electrophoresis and DNA gel].

    PubMed

    Zhu, S; Gao, Y

    1998-07-01

    This thesis introduced a kind of computerized image analysis system of medical electrophoresis and DNA gel, which have a high performance/price ratio. Moreover, it gives a detailed presentation of how to eliminate the background obstruction by the conjunction of hardware and software.

  10. Aligning Goals, Assessments, and Activities: An Approach to Teaching PCR and Gel Electrophoresis

    ERIC Educational Resources Information Center

    Phillips, Allison R.; Robertson, Amber L.; Batzli, Janet; Harris, Michelle; Miller, Sarah

    2008-01-01

    Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments…

  11. Disposable pen-shaped capillary gel electrophoresis cartridge for fluorescence detection of bio-molecules

    NASA Astrophysics Data System (ADS)

    Amirkhanian, Varoujan; Tsai, Shou-Kuan

    2014-03-01

    We introduce a novel and cost-effective capillary gel electrophoresis (CGE) system utilizing disposable pen-shaped gelcartridges for highly efficient, high speed, high throughput fluorescence detection of bio-molecules. The CGE system has been integrated with dual excitation and emission optical-fibers with micro-ball end design for fluorescence detection of bio-molecules separated and detected in a disposable pen-shaped capillary gel electrophoresis cartridge. The high-performance capillary gel electrophoresis (CGE) analyzer has been optimized for glycoprotein analysis type applications. Using commercially available labeling agent such as ANTS (8-aminonapthalene-1,3,6- trisulfonate) as an indicator, the capillary gel electrophoresis-based glycan analyzer provides high detection sensitivity and high resolving power in 2-5 minutes of separations. The system can hold total of 96 samples, which can be automatically analyzed within 4-5 hours. This affordable fiber optic based fluorescence detection system provides fast run times (4 minutes vs. 20 minutes with other CE systems), provides improved peak resolution, good linear dynamic range and reproducible migration times, that can be used in laboratories for high speed glycan (N-glycan) profiling applications. The CGE-based glycan analyzer will significantly increase the pace at which glycoprotein research is performed in the labs, saving hours of preparation time and assuring accurate, consistent and economical results.

  12. A simple monolithic column electroelution for protein recovery from gel electrophoresis.

    PubMed

    Li, Guo-Qing; Shao, Jing; Guo, Chen-Gang; Dong, Jing-Yu; Fan, Liu-Yin; Cao, Cheng-Xi

    2012-11-01

    Protein recovery from gel electrophoresis plays an important role in functional genomics and proteomics but faces a series of issues (e.g., complex procedure, low recovery, long experimental time). In this study, a monolithic column electroelution (MCE) was developed for protein recovery from gel electrophoresis. With the model proteins of bovine serum albumin (BSA), hemoglobin (Hb), and myoglobin (Mb), the developed device and method were compared with common electroelution procedures in agarose gel electrophoresis (AGE). The comparative experiments revealed that (i) the protein recovery achieved with the developed device was greater than 83%, much higher than the 41% to 50% achieved with the common devices; (ii) the running time to obtain 70% recovery was approximately 15 min, evidently shorter than the 240 min with the common devices; and (iii) the device and procedure were simple and less time-consuming as compared with those of the common devices. It was observed that the serum protein bands cut from polyacrylamide gel electrophoresis could be transferred into solution in 15 to 30 min with 82% yield. The device, along with its relevant procedure, has potential use in protein extraction and proteomics as well as in DNA studies.

  13. Beverage-Agarose Gel Electrophoresis: An Inquiry-Based Laboratory Exercise with Virtual Adaptation

    ERIC Educational Resources Information Center

    Cunningham, Steven C.; McNear, Brad; Pearlman, Rebecca S.; Kern, Scott E.

    2006-01-01

    A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As…

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

    PubMed

    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. Separation of Recombinant Therapeutic Proteins Using Capillary Gel Electrophoresis and Capillary Isoelectric Focusing.

    PubMed

    De Jong, Caitlyn A G; Risley, Jessica; Lee, Alexis K; Zhao, Shuai Sherry; Chen, David D Y

    2016-01-01

    Detailed step-by-step methods for protein separation techniques based on capillary electrophoresis (CE) are described in this chapter. Focus is placed on two techniques, capillary gel electrophoresis (CGE) and capillary isoelectric focusing (cIEF). CGE is essentially gel electrophoresis, performed in a capillary, where a hydrogel is used as a sieving matrix to separate proteins or peptides based on size. cIEF separates proteins or peptides based on their isoelectric point (pI), the pH at which the protein or peptide bears no charges. Detailed protocols and steps (including capillary preparation, sample preparation, CE separation conditions, and detection) for both CGE and cIEF presented so that readers can follow the described methods in their own labs.

  16. Ag-nanoparticle fractionation by low melting point agarose gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Guarrotxena, Nekane; Braun, Gary

    2012-10-01

    The separation of surface-enhanced raman scattering (SERS)-active Ag-multi-nanoparticle (NP) assemblies by low melting point agarose gel electrophoresis was accomplished here by controlling surface charge using NP capping agents, and the pore size of agarose gel matrix. Detailed transmission electron microscopy analysis of excised gel fractions showed dimers and small clusters to have the greatest SERS activity and a mobility in between the monomers and large aggregates. This strategy enables one to: (1) stabilize small multispherical Ag clusters against further aggregation during purification; (2) fractionate and recover spherical assemblies by nuclearity; and (3) analyze SERS-enhancements for each fraction to optimize purification conditions.

  17. Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans.

    PubMed

    John, N E; Andersen, H U; Fey, S J; Larsen, P M; Roepstorff, P; Larsen, M R; Pociot, F; Karlsen, A E; Nerup, J; Green, I C; Mandrup-Poulsen, T

    2000-11-01

    Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.

  18. Tris-acetate polyacrylamide gradient gel electrophoresis for the analysis of protein oligomerization.

    PubMed

    Cubillos-Rojas, Monica; Schneider, Taiane; Sánchez-Tena, Susana; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2016-02-01

    Here we report a new approach for studying protein oligomerization in cells using a single electrophoresis gel. We combined the use of a crosslinking reagent for sample preparation, such as glutaraldehyde, with the analysis of oligomers by Tris-acetate polyacrylamide gel electrophoresis. The use of a 3-15% Tris-acetate polyacrylamide gradient gel allows for the simultaneous analysis of proteins of masses ranging from 10 to 500 kDa. We showed the usefulness of this method for analyzing endogenous p53 oligomerization with high resolution and sensitivity in human cells. Oligomerization analysis was dependent on the crosslinker concentration used. We also showed that this method could be used to study the regulation of oligomerization. In all experiments, Tris-acetate polyacrylamide gel electrophoresis proved to be a robust, manageable, and cost- and time-efficient method that provided excellent results using a single gel. This approach can be easily extrapolated to the study of other oligomers. All of these features make this method a highly useful tool for the analysis of protein oligomerization.

  19. Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide.

    PubMed

    Li, Jialiang; Yang, Yushi; Mao, Zhou; Huang, Wenjie; Qiu, Tong; Wu, Qingzhi

    2016-12-01

    In this work, a novel agarose gel electrophoresis strategy has been developed for separation of DNA fragments by doping graphene oxide (GO) into agarose gel. The results show that the addition of GO into agarose gel significantly improved the separation resolution of DNA fragments by increasing the shift distances of both the single DNA fragments and the adjacent DNA fragments and completely eliminating the background noise derived from the diffusion of the excessive ethidium bromide (EB) dye in the gel after electrophoresis. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO. These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules.

  20. Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Li, Jialiang; Yang, Yushi; Mao, Zhou; Huang, Wenjie; Qiu, Tong; Wu, Qingzhi

    2016-09-01

    In this work, a novel agarose gel electrophoresis strategy has been developed for separation of DNA fragments by doping graphene oxide (GO) into agarose gel. The results show that the addition of GO into agarose gel significantly improved the separation resolution of DNA fragments by increasing the shift distances of both the single DNA fragments and the adjacent DNA fragments and completely eliminating the background noise derived from the diffusion of the excessive ethidium bromide (EB) dye in the gel after electrophoresis. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO. These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules.

  1. Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

    SciTech Connect

    Smith, S.B.; Bustamante, C. ); Heller, C. )

    1991-05-28

    A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.

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

    PubMed

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

    2014-09-01

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

  3. A random-walk model for retardation of interacting species during gel electrophoresis: implications for gel-shift assays.

    PubMed Central

    Belotserkovskii, B P; Johnston, B H

    1997-01-01

    We recently showed that intermolecular DNA triplexes can form during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. We proposed a model to account for the resulting apparent comigration of triplexes with the duplex band when the lifetime of the triplex is much shorter than the time of electrophoresis. The model predicts that short-lived complexes can be detected by a gel-shift assay if the faster migrating component of the complex is labeled, a slower migrating component is in excess, and the complex itself migrates more slowly than either of the components. In this case the labeled component, after dissociation from the complex, overtakes a slower migrating band of the free, unlabeled second component and can be captured by the unlabeled component and again retarded; after dissociation of the newly formed complex the cycle is repeated. If the concentration of unlabeled component in the band is larger than some critical value (c(cr)), most of the labeled component becomes trapped in this band during the entire time of gel electrophoresis, thus effectively comigrating with the slower migrating unlabeled component. We call this mechanism of comigration "cyclic capture and dissociation" (CCD). Here we present a quantitative analysis of the model of CCD comigration which predicts that CCD comigration can be used not only for the detection of relatively short-lived complexes, but also for estimation of the specificity of complex formation. Images FIGURE 1 FIGURE 4 PMID:9284297

  4. Using in situ rheology to characterize the microstructure in photopolymerized polyacrylamide gels for DNA electrophoresis.

    PubMed

    Wang, Jian; Ugaz, Victor M

    2006-09-01

    Photopolymerized cross-linked polyacrylamide hydrogels are attractive sieving matrix formulations for DNA electrophoresis owing to their rapid polymerization times and the potential to locally tailor the gel pore structure through spatial variation of illumination intensity. This capability is especially important in microfluidic systems, where photopolymerization allows gel matrices to be precisely positioned within complex microchannel networks. Separation performance is also directly related to the nanoscale gel pore structure, which is in turn strongly influenced by polymerization kinetics. Unfortunately, detailed studies of the interplay among polymerization kinetics, mechanical properties, and structural morphology are lacking in photopolymerized hydrogel systems. In this paper, we address this issue by performing a series of in situ dynamic small-amplitude oscillatory shear measurements during photopolymerization of cross-linked polyacrylamide electrophoresis gels to investigate the relationship between rheology and parameters associated with the gelation environment including UV intensity, monomer and cross-linker composition, and reaction temperature. In general, we find that the storage modulus G' increases with increasing initial monomer concentration, cross-linker concentration, and polymerization temperature. The steady-state value of G', however, exhibits a more complex dependence on UV intensity that varies with gel concentration. A simple model based on rubber elasticity theory is used to obtain estimates of the average gel pore size that are in surprisingly good agreement with corresponding data obtained from analysis of DNA electrophoretic mobility in gels cast under identical polymerization conditions.

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

    PubMed

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

    2006-08-28

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. A computerized methodology for improved virus typing by PCR-RFLP gel electrophoresis.

    PubMed

    Maramis, Christos F; Delopoulos, Anastasios N; Lambropoulos, Alexandros F

    2011-08-01

    The analysis of digitized images from polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP)gel electrophoresis examinations is a popular method for virus typing, i.e., for identifying the virus type(s) that have infected an investigated biological sample. However, being mostly manual, the conventional virus typing protocol remains laborious, time consuming, and error prone. In order to overcome these shortcomings,we propose a computerized methodology for improving virus typing via PCR-RFLP gel electrophoresis. A novel realistic observation model of the viral DNA motion on the gel matrix is employed to assist in exploiting additional virus-related information in comparison to the conventional approaches. The extracted rich information is fed to a novel typing algorithm, resulting in faster and more accurate decisions. The proposed methodology is evaluated for the case of the human papillomavirus typing on a dataset of 80 real and 1500 simulated samples, producing very satisfactory results.Ind

  8. Pulsed field electrophoresis for the separation of protein-sodium dodecyl sulfate-complexes in polyacrylamide gels.

    PubMed

    Houri, A; Starita-Geribaldi, M

    1994-01-01

    Polyacrylamide gel electrophoresis of proteins was studied using a pulsed-current mode. A new "local field" distribution was used to correct the gel patterns and optimize migration. A corrective field was applied at fixed 2 s intervals to a constant field, inducing a complex relaxation mechanism. Calculated variations in the local field directions decreased the electric strain on the gel during the run, with resultant optimum gel structure. The relaxation mechanism was found to enhance the absolute mobility of proteins with shorter running times compared to constant field gel electrophoresis (CFGE) and other pulsed field techniques. The enhancement of molecular mobility was explored by transverse pore gradient gel electrophoresis. Ferguson curves which exhibited a convex shape in CFGE were linearized by the new pulsed-field method named pulsed oscillatory high-performance electrophoresis (POPE).

  9. Transient association of the DNA-ligand complex during gel electrophoresis.

    PubMed

    Protozanova, E; Macgregor, R B

    1999-07-01

    DNA frayed wires are extremely stable multistranded complexes arising from the association of oligonucleotides with long terminal runs of consecutive guanines. Frayed wires originating from d(A15G15) have multiple binding sites for short complementary oligonucleotides such as dT10. We examine unusual band patterns obtained when complexes formed between dT10 and DNA frayed wires are resolved on nondenaturing polyacrylamide gels. Since the lifetime of the dT10-frayed wire complexes is shorter than the time of the gel run, the interaction between the components during the gel electrophoresis affects their band patterns. We have conducted chasing experiments to show that (i) the binding of dT10 to the frayed wires can occur during gel electrophoresis, and (ii) dissociation of the complexes occurs during the gel run. Rapid repetitive dissociation-reassociation of the complexes leads to a constant partitioning of dT10 between their binding sites within frayed wires. Consequently, complexes composed of frayed wires and various numbers of bound ligands appear on the gel as a single well-defined band. The mobilities of these bands decrease continuously with the concentration of the ligand reaching saturation when all the binding sites are occupied. This characteristic pattern is observed only for relatively unstable interactions. Longer ligands, i.e., oligonucleotides with higher affinity towards the binding sites, cease to exhibit the dynamic character of interaction during gel electrophoresis. These ligands form long-lived complexes with the frayed wires that appear on the gel as faint smeared bands reflecting the presence of multiple stable complexes.

  10. Antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using fully automated two-dimensional chip gel electrophoresis.

    PubMed

    Iwamoto, M; Miura, Y; Tsumoto, H; Tanaka, Y; Morisawa, H; Endo, T; Toda, T

    2014-12-01

    We here described the antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using the fully automated two-dimensional chip gel electrophoresis system (Auto2D). This system was easy and convenient to use, and the resolution obtained was more sensitive and higher than that of conventional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We separated and identified five isoforms of the 14-3-3 protein (beta/alpha, gamma, epsilon, zeta/delta, and eta) using the Auto2D system. We then examined the antioxidant effects of carnitine supplementation on the protein profiles of the cytosolic fraction in the aged rat hippocampus, demonstrating that carnitine supplementation suppressed the oxidation of methionine residues in these isoforms. Since methionine residues are easily oxidized to methionine sulfoxide, the convenient and high-resolution 2-D PAGE system can be available to analyze methionine oxidation avoiding artifactual oxidation. We showed here that the Auto2D system was a very useful tool for studying antioxidant effects through proteomic analysis of protein oxidation.

  11. A rapid 3% polyacrylamide slab gel electrophoresis method for high through put screening of LDL phenotype

    PubMed Central

    Singh, Yogendra; Lakshmy, Ramakrishnan; Gupta, Ruby; Kranthi, Vemparala

    2008-01-01

    Background Small dense LDL is reported to be associated with increased coronary artery disease risk by various epidemiological studies. The gold standard for separation and identification of LDL subtypes in plasma is ultracentrifugation which is a lengthy procedure and difficult to perform. Various other methods like NMR, HPLC, gradient gel electrophoresis (GGE) have been reported for LDL sub fractionation all of which require specialized equipments and expertise. We report here a high throughput 3% polyacrylamide slab gel electrophoresis method (PASGE) for sub fractionation of LDL which was compared with GGE, a commonly used method for LDL sub fractionation. Results The 3% PASGE method compared well with the GGE method There was a good correlation between LDL particle diameter identified by the PASGE and GGE (Pearson correlation coefficient = 0.950). A 100% concordance was found when samples were classified as per LDL phenotypes in subjects with A and B phenotype by the two methods with the concordance being 66% in subjects with intermediate (I) phenotype. The electrophoresis apparatus was optimized and designed for running twenty eight samples at a time compared to twelve to fourteen by the conventional PASGE and eight to twelve by disc electrophoresis. Conclusion The rapid 3% polyacrylamide slab gel electrphoresis method developed is simple to perform, cost-effective and can be used for the identification LDL sub fractionation and phenotyping in large epidemiological studies. PMID:19036141

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

    PubMed

    Khan, Sharik R; Kuzminov, Andrei

    2013-12-15

    Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, 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 subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal 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.

  13. On gel electrophoresis of dielectric charged particles with hydrophobic surface: A combined theoretical and numerical study.

    PubMed

    Majee, Partha Sarathi; Bhattacharyya, Somnath; Gopmandal, Partha Pratim; Ohshima, Hiroyuki

    2017-09-21

    A theoretical study on the gel electrophoresis of a charged particle incorporating the effects of dielectric polarization and surface hydrophobicity at the particle-liquid interface is made. A simplified model based on the weak applied field and low charge density assumption is also presented and compared with the full numerical model for a nonpolarizable particle to elucidate the nonlinear effects such as double layer polarization and relaxation as well as surface conduction. The main motivation of this study is to analyze the electrophoresis of the surface functionalized nanoparticle with tunable hydrophobicity or charged fluid drop in gel medium by considering the electrokinetic effects and hydrodynamic interactions between the particle and the gel medium. An effective medium approach, in which the transport in the electrolyte saturated hydrogel medium is governed by the Brinkman equation, is adopted in the present analysis. The governing electrokinetic equations based on the conservation principles is solved numerically. The Navier-slip boundary condition along with the continuity condition of dielectric displacement are imposed on the surface of the hydrophobic polarizable particle. The impact of the slip length on the electrophoresis is profound for a thinner Debye layer, however, surface conduction effect also becomes significant for a hydrophobic particle. Impact of hydrophobicity and relaxation effects are higher for a larger particle. Dielectric polarization creates a reduction in its electrophoretic propulsion and has negligible impact at the thinner Debye length as well as lower gel screening length. This article is protected by copyright. All rights reserved.

  14. Aligning Goals, Assessments, and Activities: An Approach to Teaching PCR and Gel Electrophoresis

    PubMed Central

    Robertson, Amber L.; Batzli, Janet; Harris, Michelle; Miller, Sarah

    2008-01-01

    Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments done in lab. Here we report the development and implementation of novel exercises that integrate the biological concepts of DNA structure and replication with the techniques of PCR and gel electrophoresis. Learning goals were defined based on concepts taught throughout the cell biology lab course and learning objectives specific to the PCR and gel electrophoresis lab. Exercises developed to promote critical thinking and target the underlying concepts of PCR, primer design, gel analysis, and troubleshooting were incorporated into an existing lab unit based on the detection of genetically modified organisms. Evaluative assessments for each exercise were aligned with the learning goals and used to measure student learning achievements. Our analysis found that the exercises were effective in enhancing student understanding of these concepts as shown by student performance across all learning goals. The new materials were particularly helpful in acquiring relevant knowledge, fostering critical-thinking skills, and uncovering prevalent misconceptions. PMID:18316813

  15. Peptide fractionation by SDS-free polyacrylamide gel electrophoresis for proteomic analysis via DF-PAGE.

    PubMed

    Ramos, Yassel; Besada, Vladimir; Castellanos-Serra, Lila

    2012-01-01

    Here we present a procedure for peptide fractionation by SDS-free polyacrylamide gel electrophoresis, based on discontinuous buffer systems. In the absence of SDS, peptide migration depends both on their molecular mass and on their net charge at the electrophoresis pH. By selecting the separation pH, peptide mobility is modulated. In the original discontinuous buffer system (Tris/glycine), peptides that migrate to the anode have pI values below 6.8 and distribute along the lane in a pI decreasing order, while at acidic pH, as that afforded by histidine/MOPS buffer system, peptides with pI below 5.5 are fractionated. Separation at acid pH is particularly useful for recovering phosphopeptides as well as other highly negatively charged peptides, as those containing sialic or sulfate substituents. Both separation conditions in Tris/glycine and in histidine/MOPS are applicable to proteomic studies, by dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). First, complex protein samples are separated via SDS-PAGE, and after in-gel proteolysis, peptides are loaded on a second SDS-free gel, where they are separated as described here.

  16. Aligning goals, assessments, and activities: an approach to teaching PCR and gel electrophoresis.

    PubMed

    Phillips, Allison R; Robertson, Amber L; Batzli, Janet; Harris, Michelle; Miller, Sarah

    2008-01-01

    Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments done in lab. Here we report the development and implementation of novel exercises that integrate the biological concepts of DNA structure and replication with the techniques of PCR and gel electrophoresis. Learning goals were defined based on concepts taught throughout the cell biology lab course and learning objectives specific to the PCR and gel electrophoresis lab. Exercises developed to promote critical thinking and target the underlying concepts of PCR, primer design, gel analysis, and troubleshooting were incorporated into an existing lab unit based on the detection of genetically modified organisms. Evaluative assessments for each exercise were aligned with the learning goals and used to measure student learning achievements. Our analysis found that the exercises were effective in enhancing student understanding of these concepts as shown by student performance across all learning goals. The new materials were particularly helpful in acquiring relevant knowledge, fostering critical-thinking skills, and uncovering prevalent misconceptions.

  17. Two methods that facilitate autoradiography of small /sup 32/P-labeled DNA fragments following electrophoresis in agarose gels

    SciTech Connect

    Cockerill, P.N.

    1988-02-01

    Two methods which permit detection by autoradiography of small /sup 32/P-labeled DNA fragments resolved by agarose gel electrophoresis are described. Agarose gel electrophoresis poses problems for autoradiography as (i) the gels are normally too thick to allow autoradiography without being dried first, and (ii) fragments of DNA of 1000 bp or less in length are readily lost during drying. In this study DNA fragments as small as 121 bp have been retained in agarose gels upon drying. This has been achieved by either (i) first fixing the DNA with the cationic detergent cetyltrimethylammonium bromide, or (ii) drying the agarose gels onto Zeta-Probe charge-modified membranes.

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

    PubMed Central

    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

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

    PubMed

    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.

  20. Statistical analysis of image data provided by two-dimensional gel electrophoresis for discovery proteomics.

    PubMed

    Crossett, Ben; Edwards, Alistair V G; White, Melanie Y; Cordwell, Stuart J

    2008-01-01

    Standardized methods for the solubilization of proteins prior to proteomics analyses incorporating two-dimensional gel electrophoresis (2-DE) are essential for providing reproducible data that can be subjected to rigorous statistical interrogation for comparative studies investigating disease-genesis. In this chapter, we discuss the imaging and image analysis of proteins separated by 2-DE, in the context of determining protein abundance alterations related to a change in biochemical or biophysical conditions. We then describe the principles behind 2-DE gel statistical analysis, including subtraction of background noise, spot detection, gel matching, spot quantitation for data comparison, and statistical requirements to create meaningful gel data sets. We also emphasize the need to develop reproducible and robust protocols for protein sample preparation and 2-DE itself.

  1. The quest for cross protective factors of Haemophilus parasuis using 2-D gel electrophoresis

    USDA-ARS?s Scientific Manuscript database

    In swine, Haemophilus parasuis (H. parasuis) infection causes polyserositis, arthritis, and meningitis. A range of virulent to nonvirulent strains exists between and within the 15 serovars. Because of this, the pathogenicity and subsequent protection from H. parasuis disease has yet to be elucidated...

  2. Allergome: the characterization of allergens based on a 2D gel electrophoresis approach.

    PubMed

    Chardin, Hélène; Peltre, Gabriel

    2005-10-01

    Type I hypersensitivity reactions are in constant progression in industrialized countries. The physiopathologic mechanism of these diseases implicates the production of specific immunoglobulin (Ig)E to allergenic molecules, their binding to the Fcepsilon receptor on the surface of mast cells and basophils, and the release of inflammatory mediators when allergens are introduced into the body and crosslink with the IgE bound to the cell surface. An allergen is defined as a molecule that induces the production of, and binds to, IgE. The identification of the allergenic molecules is an important goal to improve diagnosis and treatment of allergy. This characterization aims to extract proteins from the allergenic source, to analyze IgE specificity by immunoblotting and to identify the proteins that bind IgE.

  3. Quantitative polyacrylamide gel electrophoresis and specific activities of human somatotropin and its derivatives.

    PubMed

    Skyler, J S; Chrambach, A; Li, C H

    1977-04-25

    A preparation of human pituitary somatotropin examined in quantitative polyacrylamide gel electrophoresis is conformationally compact. The derived molecular weight by quantitative electrophoresis is consistant with the known mass of the hormone and value obtained by other methods. A plasmin-modified somatotropin is more compact and shows full activity in immunoassay, and in lymphocyte binding and somatotropic assays, with enhanced lactogenic activity. The N-terminal 134-amino acid fragment and a hendekakaihekaton fragment exist in non-monomeric forms. The N-terminal fragment has immunologic and biologic activity, with greatest activity in the in vivo somatotropic assay. The hendekakaihekaton fragment exhibited only marginal activity. All preparations showed heterogeneity of charge in quantitative electrophoresis with discreet charge isomerism recognizable for the native and plasmin-modified preparations.

  4. Coupling isoelectric focusing gel electrophoresis to mass spectrometry by electrostatic spray ionization.

    PubMed

    Qiao, Liang; Tobolkina, Elena; Liu, Baohong; Girault, Hubert H

    2013-05-07

    Gel electrophoresis has been used for decades as a high-resolution separation technique for proteins and protein isomers but has been limited in the coupling with MS because of low throughput and poor automaticity compared with LC-MS. In this work, we have developed an ambient ionization strategy, electrostatic spray ionization, for in situ ionization of proteins or peptides inside a surfactant-free polyacrylamide gel. The samples can be first separated by isoelectric focusing in a gel and then quickly in situ detected by scanning the gel with the electrostatic spray ionization mass spectrometry. With this strategy, nanograms of proteins or peptides inside a band are enough to be ionized for MS detection. This method for protein/peptide spots visualization is sensitive, providing sample molecular weight information while avoiding spot staining and chemical extraction procedures that can introduce contaminants and sample loss. Proof-of-principle results have demonstrated that the electrostatic spray ionization can produce sample ions from a complex background, and with a spatial resolution matching the isoelectric focusing, it is therefore a good choice to couple directly isoelectric focusing gel electrophoresis with mass spectrometry.

  5. Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci.

    PubMed

    Chen, Xiao-he; O'Dell, Sandra D; Day, Ian N M

    2002-05-01

    After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTHOI (five alleles 248-263 bp) and DYS390 (eight alleles 200-228 bp), and DYS392, a trinucleotide repeat (eight alleles 210-231 bp). A gel matrix of Duracryl, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%-1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra Green intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a Fluorlmager 595 fluorescent scanning system, and alleles were identified using Phoretix software for band migration measurement and Microsoft Excel to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian methodfor high-throughput genotyping in cohort studies, using standard laboratory equipment.

  6. Differentiation of human and animal strains of Streptococcus dysgalactiae by pulsed-field gel electrophoresis.

    PubMed

    Bert, F; Branger, C; Poutrel, B; Lambert-Zechovsky, N

    1997-05-01

    The genetic diversity among 54 human isolates and 33 animal isolates belonging to the species Streptococcus dysgalactiae (20 alpha-haemolytic Streptococcus dysgalactiae, 23 Streptococcus equisimilis, 43 group G streptococci and one group L streptococcus) was evaluated by macrorestriction analysis of chromosomal DNA with SmaI and resolution by pulsed-field gel electrophoresis. This technique revealed a high degree of intraspecies polymorphism, leading to the differentiation of 80 distinct banding patterns, and identified the presence of two major clusters, one containing isolates of human origin and the other isolates of animal origin. These results suggest than human and animal isolates of S.dysgalactiae are genetically distinct, and support the recent proposal of the subspecies S. dysgalactiae subsp. equisimilis for human isolates. The heterogeneity revealed within isolates from the same host type indicates that pulsed-field gel electrophoresis is a powerful epidemiological tool for studying S. dysgalactiae infections.

  7. Salmonella enterica Pulsed-Field Gel Electrophoresis Clusters, Minnesota, USA, 2001–2007

    PubMed Central

    Hedberg, Craig W.; Meyer, Stephanie; Boxrud, David J.; Smith, Kirk E.

    2010-01-01

    We determined characteristics of Salmonella enterica pulsed-field gel electrophoresis clusters that predict their being solved (i.e., that result in identification of a confirmed outbreak). Clusters were investigated by the Minnesota Department of Health by using a dynamic iterative model. During 2001–2007, a total of 43 (12.5%) of 344 clusters were solved. Clusters of >4 isolates were more likely to be solved than clusters of 2 isolates. Clusters in which the first 3 case isolates were received at the Minnesota Department of Health within 7 days were more likely to be solved than were clusters in which the first 3 case isolates were received over a period >14 days. If resources do not permit investigation of all S. enterica pulsed-field gel electrophoresis clusters, investigation of clusters of >4 cases and clusters in which the first 3 case isolates were received at a public health laboratory within 7 days may improve outbreak investigations. PMID:21029524

  8. Detection of connexins in liver cells using sodiumdodecylsulfate polyacrylamide gel electrophoresis and immunoblot analysis

    PubMed Central

    Willebrords, Joost; Maes, Michaël; Yanguas, Sara Crespo; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Since connexin expression is partly regulated at the protein level, immunoblot analysis represents a frequently addressed technique in the connexin research field. The present chapter describes the set-up of an immunoblot procedure, including protein extraction and quantification from biological samples, gel electrophoresis, protein transfer and immunoblotting, which is optimized for analysis of connexins in liver tissue. In essence, proteins are separated on a polyacrylamide gel using sodiumdodecylsulfate followed by transfer of proteins on a nitrocellulose membrane. The latter allows specific detection of connexins with antibodies combined with revelation through enhanced chemiluminescence. PMID:27207285

  9. Protein differences between normal and oligospermic human sperm demonstrated by two-dimensional gel electrophoresis.

    PubMed

    Morgentaler, A; Schopperle, W M; Crocker, R H; DeWolf, W C

    1990-11-01

    Protein expression by sperm obtained from men with normal semen analysis and men with oligospermia were evaluated by two-dimensional gel electrophoresis. Proteins were solubilized in a 9.5 M urea/2% Nonidet-P40 (LKB, Bromma, Sweden) lysis buffer and underwent second dimension separation on 10 to 16% polyacrylamide gradient gels. A set of 36 invariant proteins was identified in all normospermic samples, whereas 8 of 10 evaluable oligospermic samples lacked 1 or more of the invariant proteins. Proteins absent in oligospermic samples may be critical to normal sperm function and may serve as markers for infertility.

  10. Molecular characterization of Clostridium tetani strains by pulsed-field gel electrophoresis and colony PCR.

    PubMed

    Plourde-Owobi, Lucile; Seguin, Delphine; Baudin, Marie-Anne; Moste, Catherine; Rokbi, Bachra

    2005-09-01

    Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.

  11. Molecular Characterization of Clostridium tetani Strains by Pulsed-Field Gel Electrophoresis and Colony PCR

    PubMed Central

    Plourde-Owobi, Lucile; Seguin, Delphine; Baudin, Marie-Anne; Moste, Catherine; Rokbi, Bachra

    2005-01-01

    Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene. PMID:16151158

  12. Instability in the dynamics of long DNA undergoing gel electrophoresis in a strong field

    NASA Astrophysics Data System (ADS)

    Semenov, A. N.; Subbotin, A. V.

    1995-05-01

    We present a model describing reptation motion of a long DNA chain undergoing gel electrophoresis. Internal modes of the chain in an effective tube are associated with lateral loops (hernias), and the effect of the field on these lateral loops is analyzed. An equation describing reptation dynamics of the chain is derived. It is shown for fields exceeding some critical value that stationary reptation motion becomes unstable. Mobility of the chain in the weak fields is also calculated.

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

    PubMed

    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.

  14. Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis.

    PubMed

    Cavani, Luciano; Ciavatta, Claudio; Trubetskaya, Olga E; Reznikova, Olga I; Afanas'eva, Gaida V; Trubetskoj, Oleg A

    2003-01-03

    Capillary zone electrophoresis (CZE) was used for characterisation of soil humic acid (HA) fractions obtained by coupling size-exclusion chromatography with polyacrylamide gel electrophoresis, on the basis of their molecular size and electrophoretic mobility. CZE was conducted using several low alkaline buffers as background electrolyte (BGE): 50 mM carbonate, pH 9.0; 50 mM phosphate, pH 8.5; 50 mM borate, pH 8.3; 50 mM Tris-borate+1 mM EDTA+7 M urea+0.1% sodium dodecyl sulphate (SDS), pH 8.3. Independently of BGE conditions, the effective electrophoretic mobility of HA fractions were in good agreement with their molecular size. The better resolution of HA were obtained in Tris-borate-EDTA buffer with urea and SDS. This results indicated that CZE, mostly with BGE-contained disaggregating agents, is useful for separating HAs in fractions with different molecular sizes.

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

    PubMed Central

    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

  16. Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

    PubMed

    Shi, Liang; Khandurina, Julia; Ronai, Zsolt; Li, Bi-Yu; Kwan, Wai King; Wang, Xun; Guttman, András

    2003-01-01

    A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

  17. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    SciTech Connect

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-08-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application.

  18. Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Cardozo, J A; Fernández-Juan, M; Forcada, F; Abecia, A; Muiño-Blanco, T; Cebrián-Pérez, J A

    2006-09-01

    This study was conducted to evaluate monthly changes in the ram seminal plasma protein profile using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with a polyacrylamide linear gradient gel. Likewise, comparative analyses of the protein composition of ovine seminal plasma (SP) from ejaculates obtained along the year, and its relationship with sperm motility, viability and concentration of ejaculate were carried out. Western-blot analysis was performed to specifically detect P14, a ram SP protein postulated to be involved in sperm capacitation and gamete interaction [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49], and its variations along the year have also been established. The experiment was carried out from May 2003 to April 2004, with nine Rasa Aragonesa rams. Ejaculates obtained every 2 days were pooled and used for each assay, to avoid individual differences, and three two-dimensional SDS-PAGE gels were run for each month. The high resolution of the gradient gel allowed the image analysis software to detect around 252 protein spots, with pIs ranging from 4.2 to 7.6, and molecular weight (M(r)) from 12.5 to 83.9 kDa. Four protein spots (1, 2, 3 and 4) of low M(r) (15.1, 15.7, 15.9 and 21.0 kDa) and acidic pI (5.9, 5.3, 5.7 and 6.6), respectively, had the highest relative intensity in the SP map (11.2, 9.3, 4.7 and 7.7%, respectively). Spot 3 was more abundant (P<0.05) from May to December, and negatively correlated (P<0.05, r=-0.34) with sperm viability and concentration (P<0.05, r=0.36). Another 12 protein spots also had significant quantitative differences (P<0.05) along the year, and 17 protein spots, which correlated with some seminal quality parameter, did not show quantitative monthly changes. Western-blot analysis indicated that spots 1 and 2 reacted

  19. Comparative fluorescence two-dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on-gel generation of a reference protein spot grid.

    PubMed

    Ackermann, Doreen; Wang, Weiqun; Streipert, Benjamin; Geib, Birgit; Grün, Lothar; König, Simone

    2012-05-01

    The comparison of proteins separated on 2DE is difficult due to gel-to-gel variability. Here, a method named comparative fluorescence gel electrophoresis (CoFGE) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V-shaped wells are placed in a stacking gel above the pI strip. Proteins separated on the pI strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V-wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2DE protein gels, because they are generated in combination with regular in-gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using Escherichia coli lysate. For a set of 47 well-defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of pI-strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2DE-gels produced over several years and in different laboratories.

  20. Accurate quantification of DNA methylation of DRD4 applying capillary gel electrophoresis with LIF detection.

    PubMed

    Goedecke, Simon; Schlosser, Sabrina; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

    2009-04-01

    Aberrant DNA methylation of gene promoters may be investigated by an array of different technologies. Besides DNA sequencing techniques following bisulfite treatment and determination of overall methylation by quantification of 5-methylcytosine/cytosine ratio following DNA hydrolysis, most approaches rely on PCR amplification of a defined template and subsequent analysis by conventional gel electrophoresis. As an additional analytical tool, a capillary gel electrophoresis method has been developed to quantify the methylation in combined bisulfite restriction analysis products of the gene dopamine receptor D4 (DRD4). Analyses were carried out in a bare fused-silica capillary dynamically coated with a 1% w/w solution of PVA (M(r)=72,000). A buffer (pH 7.3) containing 3% w/w 2-hydroxyethylcellulose (M(nu) approximately 90,000 g/mol) was used as sieving matrix. With 1/x weighted regression the accuracy (bias) of the method is within +/-10% and the precision (expressed as RSD) also meets the common acceptance criteria of 15% (20% near lower LOQ). It overcomes the limitations of standard gel electrophoresis, which allows only one single run per analysis and requires large amounts of DNA. Therefore, the method represents a valuable tool for routine quantitative analysis of the methylation status of DRD4 and other target genes.

  1. An Inexpensive Gel Electrophoresis-Based Polymerase Chain Reaction Method for Quantifying mRNA Levels

    PubMed Central

    2005-01-01

    In order to engage their students in a core methodology of the new genomics era, an ever-increasing number of faculty at primarily undergraduate institutions are gaining access to microarray technology. Their students are conducting successful microarray experiments designed to address a variety of interesting questions. A next step in these teaching and research laboratory projects is often validation of the microarray data for individual selected genes. In the research community, this usually involves the use of real-time polymerase chain reaction (PCR), a technology that requires instrumentation and reagents that are prohibitively expensive for most undergraduate institutions. The results of a survey of faculty teaching undergraduates in classroom and research settings indicate a clear need for an alternative approach. We sought to develop an inexpensive and student-friendly gel electrophoresis-based PCR method for quantifying messenger RNA (mRNA) levels using undergraduate researchers as models for students in teaching and research laboratories. We compared the results for three selected genes measured by microarray analysis, real-time PCR, and the gel electrophoresis-based method. The data support the use of the gel electrophoresis-based method as an inexpensive, convenient, yet reliable alternative for quantifying mRNA levels in undergraduate laboratories. PMID:15917874

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

    PubMed

    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.

  3. Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan.

    PubMed

    Cowman, Mary K; Chen, Cherry C; Pandya, Monika; Yuan, Han; Ramkishun, Dianne; LoBello, Jaclyn; Bhilocha, Shardul; Russell-Puleri, Sparkle; Skendaj, Eraldi; Mijovic, Jovan; Jing, Wei

    2011-10-01

    The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100°C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.

  4. Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.

    PubMed

    Chéry, Cyrille C; Günther, Detlef; Cornelis, Rita; Vanhaecke, Frank; Moens, Luc

    2003-10-01

    The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation.

  5. DNA electrophoresis in agarose gels: A new mobility vs. DNA length dependence

    NASA Astrophysics Data System (ADS)

    Beheshti, Afshin

    2002-04-01

    Separations were performed on double stranded DNA (dsDNA) using electrophoresis. Electrophoresis is the steady transport of particles under the influence of an external electric field. Double stranded DNA fragments ranging in length from 200 base pairs (bp) to 194,000 bp (0.34 nm = 1 bp) were electrophoresed at agarose gel concentrations T = 0.4%--1.5%. The electric field was varied from 0.62 V/cm to 6.21 V/cm. A wide range of electric fields and gel concentrations were used to study the usefulness of a new interpolation equation, 1mL =1mL-( 1mL-1 ms)e-L/g , where mL,ms , and g are independent free fitting parameters. The long length mobility limit is interpreted as mL , the short length mobility limit is ms , and g is the crossover between the long length limit and the short length limit. This exponential relation fit very well (chi2 ≥ 0.999) when there are two smooth transitions observed in the "reptation plots" (plotting 3mL/m∘ vs. L) (J. Rousseau, G. Drouin, and G. W. Slater, Phys Rev Lett. 1997, 79, 1945--1948). Fits deviate from the data when three different slopes were observed in the reptation plots. Reptation plots were used to determine a phase diagram for dsDNA migration regimes. The phase diagrams define different regions where mechanisms for molecular transport affect the migration of dsDNA in agarose gels during electrophoresis. The parameters from the equation have also been interpreted to provide a physical description of the structure of the agarose gel by calculating the pore sizes. The relations between the values for the pore sizes and the phase diagrams are interpreted to better understand the migration of the DNA through agarose gels.

  6. Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis

    SciTech Connect

    McGregor, David A.

    1993-07-01

    The purpose of the Human Genome Project is outlined followed by a discussion of electrophoresis in slab gels and capillaries and its application to deoxyribonucleic acid (DNA). Techniques used to modify electroosmotic flow in capillaries are addressed. Several separation and detection schemes for DNA via gel and capillary electrophoresis are described. Emphasis is placed on the elucidation of DNA fragment size in real time and shortening separation times to approximate real time monitoring. The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis are possible throughout the separation. The use of absorption removes the need for staining or radioisotope labeling thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without de-staining. The optimization of several parameters considerably reduces total analysis time. DNA from 2 kb to 850 kb can be separated in 3 hours on a 7 cm gel with interactive control of the pulse time, which is 10 times faster than the use of a constant field program. The separation of ΦX174RF DNA-HaeIII fragments is studied in a 0.5% methyl cellulose polymer solution as a function of temperature and applied voltage. The migration times decreased with both increasing temperature and increasing field strength, as expected. The relative migration rates of the fragments do not change with temperature but are affected by the applied field. Conditions were established for the separation of the 271/281 bp fragments, even without the addition of intercalating agents. At 700 V/cm and 20°C, all fragments are separated in less than 4 minutes with an average plate number of 2.5 million per meter.

  7. Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis

    PubMed Central

    Bender, Robert A.; Streicher, Stanley L.

    1979-01-01

    We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GSE) and Klebsiella aerogenes (GSK). In gels containing sodium dodecyl sulfate (SDS), we found that GSK had a mobility which differed significantly from that of GSE. In addition, for both GSK and GSE, adenylylated subunits (GSK-adenosine 5′-monophosphate [AMP] and GSE-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GSK-AMP < GSK < GSE-AMP < GSE. We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GSK, and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GSK or GSK-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele. Images PMID:33958

  8. An evaluation of the SPIFE 3000 semi-automated gel electrophoresis system for the identification of hemoglobin variants and comparison of relative electrophoretic mobilities with manual gel electrophoresis methods.

    PubMed

    Hoyer, J D; Markley, K M; Savedra, M E; Kubik, K S; Scheidt, R M

    2010-06-01

    Laboratory identification of hemoglobin (Hb) variants can involve multiple techniques. The use of semi-automated instruments that perform gel electrophoresis and staining, such as the SPIFE 3000 electrophoresis system, can greatly reduce the labor required for these commonly used techniques. We performed a comparison of the method involved in SPIFE 3000 system with those of manual gel electrophoresis. A total of 22 540 samples were analyzed using the SPIFE 3000, and compared with mobilities on cellulose acetate and citrate agar gels using standard manual methods. The results were compared using relative electrophoretic mobilities (REM). Of the 191 Hb variants identified, only 13 had REM that differed from manual electrophoresis when analyzed using the SPIFE 3000 system. One variant (Hb O-Indonesia) showed different mobility on both acid and alkaline gels, two (Hb E, Hb Sunshine Seth) on alkaline gel only, and 10 (Hbs N-Baltimore, N-Seattle, O-Arab, Shelby, Summer Hill, Tak, Hasharon, M-Iwate, Q-Iran, and Setif) on acid gels only. The SPIFE 3000 semi-automated electrophoresis system produces similar results when compared with those of standard manual electrophoresis methods.

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

    PubMed

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

    2012-02-01

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

  10. Cytoplasmic polyhedrosis virus classification by electropherotype; validation by serological analyses and agarose gel electrophoresis.

    PubMed

    Mertens, P P; Crook, N E; Rubinstein, R; Pedley, S; Payne, C C

    1989-01-01

    Serological analyses of several different cytoplasmic polyhedrosis viruses (CPVs), including two type 1 CPVs from Bombyx mori, type 1 CPV from Dendrolimus spectabilis, type 12 CPV from Autographa gamma, type 2 CPV from Inachis io, type 5 CPV from Orgyia pseudotsugata and type 5 CPV from Heliothis armigera, demonstrated a close correlation between the antigenic properties of the polyhedrin or virus particle structural proteins and the genomic dsRNA electropherotypes. The dsRNAs of these viruses were analysed by electrophoresis in 3% and 10% polyacrylamide gels with a discontinuous Tris-HCl/Tris-glycine buffer system or by 1% agarose gel electrophoresis using a continuous Tris-acetate-EDTA buffer system. Electrophoretic analysis in agarose gels was found to be the most suitable for the classification of CPV isolates into electropherotypes, and the results obtained showed a close correlation with the observed antigenic relationships between different virus isolates. However, electrophoretic analysis in 10% polyacrylamide gels was most sensitive for the detection of intra-type variation and the presence of mixed virus isolates.

  11. A Sol-Gel-Modified Poly(methyl methacrylate) Electrophoresis Microchip with a Hydrophilic Channel Wall

    SciTech Connect

    Chen, Gang; Xu, Xuejiao; Lin, Yuehe; Wang, Joseph

    2007-07-27

    A sol-gel method was employed to fabricate a poly(methyl methacrylate) (PMMA) electrophoresis microchip that contains a hydrophilic channel wall. To fabricate such a device, tetraethoxysilane (TEOS) was injected into the PMMA channel and was allowed to diffuse into the surface layer for 24 h. After removing the excess TEOS, the channel was filled with an acidic solution for 3 h. Subsequently, the channel was flushed with water and was pretreated in an oven to obtain a sol-gel-modified PMMA microchip. The water contact angle for the sol-gel-modified PMMA was 27.4° compared with 66.3° for the pure PMMA. In addition, the electro-osmotic flow increased from 2.13×10-4 cm2 V-1 s-1 for the native-PMMA channel to 4.86×10-4 cm2 V-1 s-1 for the modified one. The analytical performance of the sol-gel-modified PMMA microchip was demonstrated for the electrophoretic separation of several purines, coupled with amperometric detection. The separation efficiency of uric acid increased to 74 882.3 m-1 compared with 14 730.5 m-1 for native-PMMA microchips. The result of this simple modification is a significant improvement in the performance of PMMA for microchip electrophoresis and microfluidic applications.

  12. Band broadening in gel electrophoresis: scaling laws for the dispersion coefficient measured by FRAP.

    PubMed

    Tinland, B; Pernodet, N; Pluen, A

    1998-10-05

    We determined quantitatively the band broadening effect during gel electrophoresis by measuring the longitudinal dispersion coefficient Dx, with a fluorescence recovery after photobleaching setup, coupled to an electrophoretic cell. We carried out measurements as a function of the electric field, the average pore size, and the molecular length of DNA fragments. Our results are in good agreement with the predictions of the biased reptation model with fluctuations described by T. A. Duke et al. [(1992) Physics Review Letters, vol. 69, pp. 3260-3263]. This agreement is observed on single-stranded DNA [persistence length approximately equal to 4 nm; B. Tinland et al. (1997) Macromolecules, vol. 30, pp. 5763-5765] in polyacrylamide gels and on double-stranded DNA (persistence length approximately equal to 50 nm) in agarose gels, two systems where the ratio between the average pore size and the Kuhn length is larger than 1.

  13. Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data

    NASA Astrophysics Data System (ADS)

    Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.

    2008-01-01

    We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

  14. Gel electrophoresis of DNA partially denatured at the ends: what are the dominant conformations?

    PubMed

    Sean, David; Slater, Gary W

    2013-03-01

    Gel electrophoresis of a partially denatured dsDNA fragment is studied using Langevin Dynamics computer simulations. For simplicity, the denatured ssDNA sections are placed at the ends of the fragment in a symmetrical fashion. A squid-like conformation is found to sometimes cause the fragment to completely block in the gel. In fact, this conformation is the principal cause of the steep reduction in mobility observed in the simulations. As the field is increased, it is found that the occurrence of this conformation dominates the migration dynamics. Although the squid conformation seems to be more stable at high fields, the field can eventually force the fragments to thread through the gel pores regardless. We qualitatively explore the behavior of this squid-like conformation across a range of fields and degrees of denaturation, and we discuss the relevance of our findings for TGGE.

  15. Comparative Analysis of Denaturing Gradient Gel Electrophoresis and Temporal Temperature Gradient Gel Electrophoresis Profiles as a Tool for the Differentiation of Candida Species.

    PubMed

    Mohammadi, Parisa; Hamidkhani, Aida; Asgarani, Ezat

    2015-10-01

    Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will be obtained quickly. In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their results were compared. Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions. The amplified fragments were then used to analyze DGGE and TTGE profiles. The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set. Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower costs.

  16. Comparative Analysis of Denaturing Gradient Gel Electrophoresis and Temporal Temperature Gradient Gel Electrophoresis Profiles as a Tool for the Differentiation of Candida Species

    PubMed Central

    Mohammadi, Parisa; Hamidkhani, Aida; Asgarani, Ezat

    2015-01-01

    Background: Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will be obtained quickly. Objectives: In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their results were compared. Materials and Methods: Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions. The amplified fragments were then used to analyze DGGE and TTGE profiles. Results: The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set. Conclusions: Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower costs. PMID:26568801

  17. Sol-gel chemistry-based Ucon-coated columns for capillary electrophoresis.

    PubMed

    Hayes, J D; Malik, A

    1997-07-18

    A sol-gel chemistry-based novel approach for the preparation of a Ucon-coated fused-silica capillary column in capillary electrophoresis is presented. In this approach the sol-gel process is carried out inside 25 microm I.D. fused-silica capillaries. The sol solution contained appropriate quantities of an alkoxide-based sol-gel precursor, a polymeric coating material (Ucon), a crosslinking reagent, a surface derivatizing reagent, controlled amounts of water and a catalyst dissolved in a suitable solvent system. The coating procedure involves filling a capillary with the sol solution and allowing the sol-gel process to proceed for an optimum period. Hydrolysis of the alkoxide precursor and polycondensation of the hydrolyzed products with the surface silanol groups and the hydroxy-terminated Ucon molecules lead to the formation of a surface-bonded sol-gel coating on the inner walls of the capillary. The thickness of the coated film can be controlled by varying the reaction time, coating solution composition and experimental conditions. Commercial availability of high purity sol-gel precursors (e.g., TEOS 99.999%), the ease of coating, run-to-run and column-to-column reproducibility, and long column lifetimes make sol-gel coating chemistry very much suitable for being applied in analytical microseparations column technology. Test samples of basic proteins and nucleotides were used to evaluate the column performance. These results show that the sol-gel coating scheme has allowed for the generation of bio-compatible surfaces characterized by high separation efficiencies in CE. For different types of solutes, the sol-gel coated Ucon column consistently provided migration time R.S.D. values of the order of 0.5%.

  18. Establishment of two-dimensional gel electrophoresis profiles of the human acute promyelocytic leukemia cell line NB4.

    PubMed

    He, Pengcheng; Liu, Yanfeng; Zhang, Mei; Wang, Xiaoning; Wang, Huaiyu; Xi, Jieying; Wei, Kaihua; Wang, Hongli; Zhao, Jing

    2012-09-01

    To explore optimum conditions for establishing a two‑dimensional gel electrophoresis (2-DE) map of the human acute promyelocytic leukemia (APL) cell line NB4 and to analyze its protein profiles, we extracted total proteins from NB4 cells using cell disruption, liquid nitrogen freeze-thawing and fracturing by ultrasound, and quantified the extracted protein samples using Bradford's method. 2-DE was applied to separate the proteins, which were silver-stained in the gel. Well‑separated protein spots were selected from the gel using the ImageMaster™ 2D Platinum analysis system. Moreover, the effects of various protein sample sizes (140, 160 and 180 µg) on the 2-DE maps of the NB4 cells were determined and compared. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), peptide mass fingerprinting (PMF) and database searching were used to identify the proteins. When the quantity of loading proteins was 160 µg, clear, well-resolved, reproducible 2-DE proteomic profiles of the NB4 cells were obtained. The average number of protein spots in 3 gels was 1160±51 with an average matching rate of 81%. A total of 10 proteins were identified by mass spectrometry and database queries, certain proteins were products of oncogenes and others were involved in cell cycle regulation and signal transduction. In summary, 2-DE profiles of the proteome of NB4 cells were established and certain proteins were identified by MALDI-TOF-MS and PMF which lay the foundation of further proteomic research of NB4 cells. These data should be useful for establishing a human APL proteome database.

  19. The Sensitivity of Gel Electrophoresis as a Detector of Genetic Variation

    PubMed Central

    Ramshaw, John A. M.; Coyne, Jerry A.; Lewontin, R. C.

    1979-01-01

    Three experiments based on an idea of Youderian have been performed to determine the proportions and kinds of amino acid substitutions that are detected by gel electrophoresis when applied to surveys of protein variation in populations. The experiments involved applying the sequential method of electrophoresis under several conditions of pH and gel concentration to a large sample of human hemoglobins with known amino acid substitutions. In the first experiment, a random sample of 20 different hemoglobin variants was studied, and these were separated into 17 distinct electrophoretic classes by three sequential gel conditions, thus giving a detectability of 85%. A single pass under standard conditions detected eight classes. The second experiment compared groups of substitutions that were chemically identical, but in different positions in the α and β chains, while the third experiment compared pairs of substitutions that were charge equivalent, but chemically different at the same chain position. The sequential method distinguished 90% of all chemically identical substitutions when they were at different chain locations, and four out of five charge equivalent but chemically different substitutions at the same site. Examination of the location of each substitution in the three-dimensional structure of hemoglobins showed that interior substitutions usually are less different from Hb A than are surface substitutions and that local interactions with chain and spatial neighbors are sufficient to distinguish substitutions in very similar positions on the outside of the molecule. The "charge ladder" model of electrophoretic classes is clearly incorrect, and it appears that sequential gel electrophoresis as practiced in our Drosophila surveys has detected a substantial fraction of amino acid substitutions if hemoglobin is regarded as a model. This estimate may be modified as other molecules beside hemoglobin are subjected to similar calibration experiments. PMID:546674

  20. Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis

    SciTech Connect

    Kogan, S.; Gitschier, J. )

    1990-03-01

    Hemophilia A results from mutations in the gene coding for coagulation factor VIII. The authors gradient gel electrophoresis to screen for mutations in the region of the factor VIII gene coding for the first acidic domain. Amplification primers were designed employing the MELTMAP computer program to optimize the ability to detect mutations. Screening of amplified DNA from 228 unselected hemophilia A patients revealed two mutations and one polymorphism. Rescreening the same population by making heteroduplexes between amplified patient and control samples prior to electrophoresis revealed one additional mutation. The mutations include two missense and one 4-base-pair deletion, and each mutation was found in patients with severe hemophilia. The polymorphism, located adjacent to the adenine branch site in intron 7, is useful for genetic prediction in some cases where the Bcl I and Xba I polymorphisms are uninformative. These results suggest that DNA amplification and denaturing gradient gel electrophoresis should be an excellent strategy for identifying mutations and polymorphisms in defined regions of the factor VIII gene and other large genes.

  1. Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis.

    PubMed

    Shang, Ying; Zhu, Pengyu; Xu, Wentao; Guo, Tianxiao; Tian, Wenying; Luo, Yunbo; Huang, Kunlun

    2013-12-15

    In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5' or 3' end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1 ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP-MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops.

  2. Detection of genotoxic insult as DNA strand breaks in fish blood cells by agarose gel electrophoresis

    SciTech Connect

    Theodorakis, C.W. ); D'Surney, S.J. . Dept. of Biology); Shugart, L.R. . Environmental Sciences Division)

    1994-07-01

    DNA, isolated from the blood cells of bluegill sunfish (Lepomis macrochirus) exposed in the lab to bedded sediment collected from a site contaminated with genotoxic compounds (i.e., PAHs, PCBs, and heavy metals), was examined for strand breakage by agarose gel electrophoresis. Before electrophoresis the blood cells were embedded in agarose plugs and incubated with proteinase. After electrophoresis under both neutral (pH 7) or alkaline (pH 12) conditions, the median molecular length (MML) of the DNA distributed in the gel was determined. These quantitative measures were used to estimate the difference in the number of double- and single-strand breaks between DNA preparations. Both types of strand breakage were found to be greater in fish exposed to sediment contaminated with genotoxic compounds as compared to nonexposed fish. A statistically significant correlation was demonstrated between the MML value obtained by the electrophoretic assay reported here and the F value (measure of DNA double-strandedness) obtained by the alkaline unwinding assay.

  3. Single nucleus versus single-cell gel electrophoresis: kinetics of DNA track formation.

    PubMed

    Afanasieva, Katerina; Chopei, Marianna; Sivolob, Andrei

    2015-04-01

    Single-cell gel electrophoresis, or the comet assay, is usually performed with nucleoids prepared after a lysis of either whole cells (more often) or isolated cell nuclei (rarely). Electrophoretic properties of the second type of nucleoids have never been investigated carefully. We measured the kinetics of the DNA exit from nuclei-derived nucleoids in comparison with cell-derived nucleoids. The results show that general organization of the nuclei-derived nucleoids is not changed very much in comparison with nucleoids commonly obtained from whole cells. At the same time, in contrast to the cell-derived nucleoids, for which the exit is stepwise and cooperative, the DNA exit from the nuclei-derived nucleoids can be described by a simple monomolecular kinetics. This difference is probably due to agarose penetration into nuclei (but not into cells) before polymerization of the agarose gel. We suggest that single-nucleus gel electrophoresis may be a way for the comet assay standardization. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

    PubMed Central

    Gardano, Laura; Holland, Linda; Oulton, Rena; Le Bihan, Thierry; Harrington, Lea

    2012-01-01

    Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. PMID:22187156

  5. Proposal for a Standard Representation of Two-Dimensional Gel Electrophoresis Data

    PubMed Central

    Wastling, Jonathan; Hunt, Ela

    2003-01-01

    The global analysis of proteins is now feasible due to improvements in techniques such as two-dimensional gel electrophoresis (2-DE), mass spectrometry, yeast two-hybrid systems and the development of bioinformatics applications. The experiments form the basis of proteomics, and present significant challenges in data analysis, storage and querying. We argue that a standard format for proteome data is required to enable the storage, exchange and subsequent re-analysis of large datasets. We describe the criteria that must be met for the development of a standard for proteomics. We have developed a model to represent data from 2-DE experiments, including difference gel electrophoresis along with image analysis and statistical analysis across multiple gels. This part of proteomics analysis is not represented in current proposals for proteomics standards. We are working with the Proteomics Standards Initiative to develop a model encompassing biological sample origin, experimental protocols, a number of separation techniques and mass spectrometry. The standard format will facilitate the development of central repositories of data, enabling results to be verified or re-analysed, and the correlation of results produced by different research groups using a variety of laboratory techniques. PMID:18628860

  6. Unhooking dynamics of U-shaped DNA molecule undergoing gel electrophoresis.

    PubMed

    Song, L; Maestre, M F

    1991-08-01

    It has been found that DNA molecules are often hooked around obstacles in a U-shaped configuration in gel electrophoresis. To understand the dynamics of the unhooking of U-shaped DNA molecules undergoing gel electrophoresis, we have examined the length changes of the longer and shorter arms of the U-shape as a function of time. Two types of unhooking have been found. In one type, the length changes of both arms are expontential in time but with different time constants. In another type, the length changes of the shorter arm is exponential and that of the longer one is linear with time. The interpretation is that the extent of stretch of the spring-like DNA chain decreases as the length difference between the two arms increases during the unhooking processes, and that the frictions at the pivot point can be relatively large depending upon the local structure of the gel. The friction coefficient at the pivot point is estimated to be nu 0 = (2.98 +/- 1.42)x10(-5) g/sec.

  7. Gel electrophoresis of a charge-regulated, bi-functional particle.

    PubMed

    Hsu, Jyh-Ping; Huang, Chih-Hua; Tseng, Shiojenn

    2013-03-01

    Adopting a Brinkman fluid model, we analyzed the electrophoresis of a charged-regulated, bi-functional particle containing both acidic and basic functional groups in a gel solution. Both the long-range hydrodynamic effect arising from the liquid drag and the short-range steric effect from particle-polymer interaction are considered. The type of particle considered is capable of simulating both biocolloids such as microorganisms and cells, and particles with adsorbed polyelectrolyte or membrane layer. Our model describes successfully the experimental data in the literature. The presence of gel has the effect of reducing the particle mobility and alleviating double-layer polarization so that the particle behavior is less complicated than that in the case where gel is absent. On the other hand, both the quantitative and qualitative behaviors of a particle depend highly on solution pH and background salt concentration, yielding interesting and significant results. These results provide valuable information for both experimental data interpretation and electrophoresis devices design.

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

    PubMed

    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.

  9. Dispersion functions and factors that determine resolution for DNA sequencing by gel electrophoresis

    SciTech Connect

    Sutherland, J.C.; Reynolds, K.J.; Fisk, D.J.

    1996-04-01

    The number of bases that can be read in a single run by a DNA sequencing instrument that detects fluorophore labeled DNA arriving at a ``finish-line`` located a fixed distance from the starting wells is influenced by numerous parameters. Strategies for improving the length-of-read of a DNA sequencer can be based on quantitative models of the separation of DNA by gel electrophoresis. The dispersion function of the electrophoretic system--the relationship between molecular contour length and time of arrival at the detector--is useful in characterizing the performance of a DNA sequencer. We adapted analytical representations of dispersion functions, originally developed for snapshot imaging of DNA gels, (samples electrophoresed for constant time), to finish-line imaging, and demonstrated that a logistic-type function with non-integral exponent is required to describe the experimental data. We use this dispersion function to determine the resolution length and resolving power of a LI-COR DNA sequencing system and a custom built capillary gel electrophoresis system, and discuss the factors that presently limit the number of bases that can be determined reliably in a single sequencing run.

  10. Measurement of DNA damage using agarose gel electrophoresis and electronic imaging

    SciTech Connect

    Sutherland, J.C.; Bergman, A.M.; Chen, Chun-Zhang; Monteleone, D.C.; Trunk, J.; Sutherland, B.M.

    1988-01-01

    Damage done to DNA by ultraviolet (uv) light, gamma rays and other carcinogens can be quantified using agarose gel electrophororesis. Agents that either produce strand breaks directly or that produce lesions that can be enzymatically or chemically converted to strand breaks can be studied. The method requires: (1) accurate measurement of the disribution of mass of DNA as a function of the distance of migration in the gel, (2) determination of the dispersion function of the electrophoresis system and (3) calculation of weighted averages of these functions by a computer. Less than 50 ng of DNA are required and the DNA need not be labeled with a radioactive tracer. Hence, the damage and repair of DNA in non-dividing cells and intact organisms---including humans---can be studied. Initial applications have focused on the quantitation of cyclobutyl pyrimidine dimers in the DNA of uv irradiated human skin. The sensitivity of lesion detection is increased by unidirectional pulsed field electrophoresis and other methods that separate longer DNA molecules. Replacing photographic detection of ethidium fluorescence by electronic imaging increases the accuracy of the measurement and the speed of data analysis. Quantitative electronic imaging of gel fluorescence offers advantages over photography in other areas of molecular biology, medicine and biotechnology. 26 refs., 5 figs.

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Accuracy of two-color peak-height-encoded DNA sequencing by capillary gel electrophoresis and laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Bay, Sue; Starke, Heather; Elliott, John; Dovichi, Norman J.

    1993-06-01

    The two-color peak-height encoded DNA sequencing technique was evaluated with six clones taken from the malaria genome. This technique produced a sequencing accuracy of at least 97.5%. Capillary gel electrophoresis, using a modest voltage of 200 V/cm, showed a three- fold increase in speed and higher efficiency compared to conventional slab gel technique.

  13. Megabase-scale mapping of the HLA gene complex by pulsed field gel electrophoresis

    SciTech Connect

    Lawrance, S.K.; Smith, C.L.; Srivastava, R.; Cantor, C.R.; Weissman, S.M.

    1987-03-13

    In the study of the genetic structure of mammalian chromosomes, there exists a resolution gap between molecular cloning experiments and meiotic linkage analyses. This gap has discouraged attempts to construct full-scale genetic maps of mammalian chromosomes. The organization of the human major histocompatibility complex was examined within this range by pulsed field gel electrophoresis. The data obtained indicate that the complex spans over 3000 kilobases and enable the construction of a megabase-scale molecular map. These results indicate that the techniques employed in DNA extraction, enzymatic digestion, electrophoresis, and hybridization are suitable for the efficient analysis of megabase regions of mammalian chromosomes and effectively bridge the resolution gap between molecular cloning and classical genetics.

  14. An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection.

    PubMed

    Chen, Xiaojia; Ugaz, Victor M

    2006-02-01

    In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.

  15. Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis.

    PubMed

    Zhou, Jianzhong; Liu, Xiaoli; Jiang, Hanhu; Dong, Mingsheng

    2009-12-01

    The microflora of Tibetan kefir grains was investigated by culture- independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rRNA for bacteria and 26S rRNA for yeasts, followed by sequencing of the most intense bands, showed that the dominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua. The bacterial communities between three kinds of Tibetan kefir grains showed 78-84% similarity, and yeasts 80-92%. The microflora is held together in the matrix of fibrillar material composed largely of a water-insoluble polysaccharide.

  16. The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Zieboll, Gerhard; Orgel, Leslie E.

    1994-01-01

    We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

  17. Genetic profiling of Klebsiella pneumoniae: comparison of pulsed field gel electrophoresis and random amplified polymorphic DNA

    PubMed Central

    Ashayeri-Panah, Mitra; Eftekhar, Fereshteh; Ghamsari, Maryam Mobarak; Parvin, Mahmood; Feizabadi, Mohammad Mehdi

    2013-01-01

    In this study, the discriminatory power of pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods for subtyping of 54 clinical isolates of Klebsiella pneumoniae were compared. All isolates were typeable by RAPD, while 3.6% of them were not typeable by PFGE. The repeatability of both typing methods were 100% with satisfying reproducibility (≥ 95%). Although the discriminatory power of PFGE was greater than RAPD, both methods showed sufficient discriminatory power (DI > 0.95) which reflects the heterogeneity among the K. pneumoniae isolates. An optimized RAPD protocol is less technically demanding and time consuming that makes it a reliable typing method and competitive with PFGE. PMID:24516423

  18. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    PubMed

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A

    2011-01-01

    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  19. TWO-DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF BROWN ALGAL PROTEIN EXTRACTS(1).

    PubMed

    Contreras, Loretto; Ritter, Andrés; Dennett, Geraldine; Boehmwald, Freddy; Guitton, Nathalie; Pineau, Charles; Moenne, Alejandra; Potin, Philippe; Correa, Juan A

    2008-10-01

    High-quality protein extracts are required for proteomic studies, a field that is poorly developed for marine macroalgae. A reliable phenol extraction protocol using Scytosiphon gracilis Kogame and Ectocarpus siliculosus (Dillwyn) Lyngb. (Phaeophyceae) as algal models resulted in high-quality protein extracts. The performance of the new protocol was tested against four methods available for vascular plants and a seaweed. The protocol, which includes an initial step to remove salts from the algal tissues, allowed the use of highly resolving two-dimensional gel electrophoresis (2-DE) protein analyses, providing the opportunity to unravel potentially novel physiological processes unique to this group of marine organisms.

  20. Application of denaturing gradient gel electrophoresis to detect DNA sequence differences encoding apolipoprotein E isoforms

    SciTech Connect

    Parker, S.; Angelico, M.C.; Laffel, L.; Krolewski, A.S. Harvard Medical School, Boston, MA )

    1993-04-01

    Apolipoprotein E (apoE) plays an important role in plasma lipid metabolism. Three common isoforms of this protein have been identified by the isoelectric focusing method. In this report the authors describe a new method for distinguishing these isoforms. Their method employs PCR amplification of the DNA sequence of exon 4 in the apoE gene followed by denaturing gradient gel electrophoresis (DGGE) to distinguish its different melting characteristics. Identification of the ApoE isoforms through DNA melting behavior rather than protein charge differences eliminates the problems associated with isoelectric focusing and facilitates screening for additional mutations at the apoE locus. 12 refs., 2 figs.

  1. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using blue native gel electrophoresis.

    PubMed

    Díaz, Francisca; Barrientos, Antoni; Fontanesi, Flavia

    2009-10-01

    The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been progressively developed. Based on methods described elsewhere, the use of blue native gel electrophoresis (BNGE) techniques to routinely assess the OXPHOS system and screen for enzymatic defects in homogenates or mitochondrial preparations from tissues or cultured cells is described here.

  2. Molecular subtyping of Clostridium botulinum by pulsed-field gel electrophoresis.

    PubMed

    Lúquez, Carolina; Joseph, Lavin A; Maslanka, Susan E

    2015-01-01

    Pulsed-field gel electrophoresis (PFGE) has been extensively used to estimate the genetic diversity of Clostridium botulinum. In addition, PFGE is the standard method for investigating foodborne outbreaks associated with various enteric pathogens, including C. botulinum. PFGE can be used to exclude a suspected but not confirmed food source when the patterns of the food and clinical isolates are different. Indistinguishable PFGE patterns may also be useful for linking isolates between patients or to a food source, but results must be interpreted within an epidemiological context to ensure isolates are truly related. Here, we describe a standardized laboratory protocol for molecular subtyping of C. botulinum by PFGE.

  3. The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Zieboll, Gerhard; Orgel, Leslie E.

    1994-01-01

    We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

  4. Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels

    SciTech Connect

    Sutherland, B.M.; Shih, A.G.

    1983-02-15

    We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs. DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels. From their migration relative to known molecular weight standards, their median molecular weight and thus the number of dimers per DNA molecule in each sample can be calculated. Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing. In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.

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

    PubMed

    Raghupathy, V; Oommen, Anna; Ramachandran, Anup

    2014-06-15

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

  6. Accommodating brightness and exposure levels in densitometry of stained polyacrylamide electrophoresis gels

    SciTech Connect

    Tan, Han Yen; Ng, Tuck Wah; Liew, Oi Wah

    2010-03-20

    Flatbed scanner densitometers can be operated under various illumination and recording exposure levels. In this work, we show that optical density measurement accuracy, sensitivity, and stability of stained polyacrylamide electrophoresis gel densitometry are crucially dependent on these two factors (brightness and exposure level), notwithstanding that the source is monochromatic, spatially uniform, and the measurements are made using an accurately calibrated step wedge in tandem. We further outline a method to accommodate the intensity deviations over a range of illumination and exposure levels in order to maintain sensitivity and repeatability in the computed optical densities. Comparisons were also made with results from a commercial densitometer.

  7. Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis.

    PubMed

    Schvartzman, J B; Martínez-Robles, M L; Hernández, P; Krimer, D B

    2010-01-01

    During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.

  8. Two Dimensional Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically-Labeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE. This provides a means of determining whether or not the biosynthetic rates of the entire granule constituents are coordinately regulated.

  9. Performing isoelectric focusing and simultaneous fractionation of proteins on a rotary valve followed by sodium dodecyl-polyacrylamide gel electrophoresis.

    PubMed

    Wang, Wei; Lu, Joann J; Gu, Congying; Zhou, Lei; Liu, Shaorong

    2013-07-16

    In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl-polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed.

  10. Characterization of immunoreactive proteins of Setaria cervi isolated by preparative polyacrylamide gel electrophoresis.

    PubMed

    Priyadarshi, Priyanka; Dravid, Piyush; Sheikh, Inayat Hussain; Saxena, Sunita; Tandon, Ashish; Kaushal, Deep C; Ali, Shakir; Kaushal, Nuzhat A

    2017-03-01

    Filarial parasites are complex mixtures of antigenic proteins and characterization of these antigenic molecules is essential to identify the diagnostically important filaria-specific antigens. In the present study, we have fractionated the somatic extracts from adults of Setaria cervi (bovine filarial parasite) on preparative SDS-polyacrylamide gel electrophoresis and tested the immunoreactivity of the separated gel fractions with polyclonal antibodies against filarial excretory-secretory antigens as well as filarial patients sera. The SDS-PAGE analysis of gel eluted fractions revealed 1 protein band in F-1 fraction, 2 protein bands in F-2 fraction and 2-3 protein bands in all other fractions (F3- F11). Seven gel eluted fractions (F1, F2, F3, F4, F5, F6 and F11) showed high ELISA reactivity with the polyclonal antibody (against excretory-secretory antigen) and four of these fractions (F-1, F-2, F3 and F6) exhibited high ELISA reactivity with antibodies present in filarial patient sera. The reactivities of the gel fractions (F1 and F2), recognized by filarial patients sera, were also tested with the monoclonal antibody (detecting the filarial circulating antigen). The F1 and F2 gel eluted fractions were found to have the target antigen of monoclonal antibody as evident by high reactivity with the monoclonal antibody in ELISA and immunoblotting. The S. cervi gel eluted F1 fraction (containing single antigen) could detect antibodies in filarial patients sera and not in non-filarial sera thereby suggesting its usefulness for specific serodiagnosis of human filariasis.

  11. Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis.

    PubMed

    Issa, Samah M A; Schulz, Benjamin L; Packer, Nicolle H; Karlsson, Niclas G

    2011-12-01

    Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.

  12. Comparison between a second generation automated multicapillary electrophoresis system with an automated agarose gel electrophoresis system for the detection of M-components.

    PubMed

    Larsson, Anders; Hansson, Lars-Olof

    2008-01-01

    During the last decade, capillary electrophoresis (CE) has emerged as an interesting alternative to traditional analysis of serum, plasma and urine proteins by agarose gel electrophoresis. Initially there was a considerable difference in resolution between the two methods but the quality of CE has improved significantly. We thus wanted to evaluate a second generation of automated multicapillary instruments (Capillarys, Sebia, Paris, France) and the high resolution (HR) buffer for serum or plasma protein analysis with an automated agarose gel electrophoresis system for the detection of M-components. The comparison between the two systems was performed with patients samples with and without M-components. The comparison included 76 serum samples with M-components > 1 g/L. There was a total agreement between the two methods for detection of these M-components. When studying samples containing oligoclonal bands/small M-components, there were differences between the two systems. The capillary electrophoresis system detected a slightly higher number of samples with oligoclonal bands but the two systems found oligoclonal bands in different samples. When looking at resolution, the agarose gel electrophoresis system yielded a slightly better resolution in the alpha and beta regions, but it required an experienced interpreter to be able to benefit from the increased resolution. The capillary electrophoresis has shorter turn-around times and bar-code reader that allows positive sample identification. The Capillarys in combination with HR buffer gives better resolution of the alpha and beta regions than the same instrument with the beta1-beta2+ buffer or the Paragon CZE2000 (Beckman) which was the first generation of capillary electrophoresis systems.

  13. Interlaboratory Agreement of Pulsed-Field Gel Electrophoresis Identification of Leptospira Serovars

    PubMed Central

    Mende, Katrin; Galloway, Renee L.; Becker, Sara J.; Beckius, Miriam L.; Murray, Clinton K.; Hospenthal, Duane R.

    2013-01-01

    Leptospirosis may be caused by > 250 Leptospira serovars. Serovar classification is a complex task that most laboratories cannot perform. We assessed the interlaboratory reproducibility of a pulsed-field gel electrophoresis (PFGE) identification technique developed by the Centers for Disease Control and Prevention (CDC). Blinded exchange of 93 Leptospiraceae strains occurred between San Antonio Military Medical Center (SAMMC) and the CDC. PFGE was performed and gel images were analyzed and compared with patterns present in each laboratory's database (CDC database: > 800 strain patterns; SAMMC database: > 300 strain patterns). Overall, 93.7% (74 of 79) of strains present in each receiving laboratory's database were correctly identified. Five isolates were misidentified, and two isolates did not match serovar PFGE patterns in the receiving laboratory's database. Patterns for these seven isolates were identical between laboratories; four serovars represented misidentified reference strains. The PFGE methodology studied showed excellent interlaboratory reproducibility, enabling standardization and data sharing between laboratories. PMID:23817329

  14. Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch.

    PubMed

    Martínez, T F; Alarcón, F J; Díaz-López, M; Moyano, F J

    2000-08-01

    An improved method, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for detection of amylase activity is described. This method will allow better characterization of certain amylases than that obtained by the Davis technique. The main features of the technique are: (i) identification of amylase bands and molecular mass determination are possible in the same gel; (ii) the hydrolysis of copolymerized substrate during electrophoretic separation is prevented using very low temperatures instead of inactivating agents such as chelating agents; and (iii) the technique is applicable to reveal amylase activity in a wide range of biological samples. The method is not useful for enzymes sensitive to SDS and for high molecular mass amylases.

  15. Interlaboratory agreement of pulsed-field gel electrophoresis identification of Leptospira serovars.

    PubMed

    Mende, Katrin; Galloway, Renee L; Becker, Sara J; Beckius, Miriam L; Murray, Clinton K; Hospenthal, Duane R

    2013-08-01

    Leptospirosis may be caused by > 250 Leptospira serovars. Serovar classification is a complex task that most laboratories cannot perform. We assessed the interlaboratory reproducibility of a pulsed-field gel electrophoresis (PFGE) identification technique developed by the Centers for Disease Control and Prevention (CDC). Blinded exchange of 93 Leptospiraceae strains occurred between San Antonio Military Medical Center (SAMMC) and the CDC. PFGE was performed and gel images were analyzed and compared with patterns present in each laboratory's database (CDC database: > 800 strain patterns; SAMMC database: > 300 strain patterns). Overall, 93.7% (74 of 79) of strains present in each receiving laboratory's database were correctly identified. Five isolates were misidentified, and two isolates did not match serovar PFGE patterns in the receiving laboratory's database. Patterns for these seven isolates were identical between laboratories; four serovars represented misidentified reference strains. The PFGE methodology studied showed excellent interlaboratory reproducibility, enabling standardization and data sharing between laboratories.

  16. Use of Two-Dimensional Polyacrylamide Gel Electrophoresis to Identify and Classify Rhizobium Strains

    PubMed Central

    Roberts, Gary P.; Leps, Walter T.; Silver, Lin E.; Brill, Winston J.

    1980-01-01

    Fifty-seven strains of various Rhizobium species were analyzed by two-dimensional gel electrophoresis. Since the protein pattern on such gels is a reflection of the genetic background of the tested strains, similarities in pattern allowed us to estimate the relatedness between these strains. All group II rhizobia (slow growing) were closely related and were very distinct from group I rhizobia (fast growing). Rhizobium meliloti strains formed a distinct group. The collection of R. leguminosarum and R. trifolii strains together formed another distinct group. Although there were some similarities within the R. phaseoli, sesbania rhizobia, and lotus rhizobia, the members within these seemed much more diverse than the members of the above groups. The technique also is useful to determine whether two unknown strains are identical. Images PMID:16345514

  17. Analysis of mitochondrial respiratory chain supercomplexes using blue native polyacrylamide gel electrophoresis (BN-PAGE)

    PubMed Central

    Jha, Pooja; Wang, Xu; Auwerx, Johan

    2016-01-01

    Mitochondria are cellular organelles that produce energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in-gel activity of SCs and the individual complexes with a good resolution on blue native polyacrylamide gel electrophoresis (BN-PAGE). PMID:26928661

  18. A model of the DNA transient orientation overshoot during gel electrophoresis

    SciTech Connect

    Lim, H.A. ); Slater, G.W.; Noolandi, J. )

    1990-01-01

    Linear dichroism and electric birefringence measurements show that when an electric field is applied to a DNA molecule at equilibrium in an agarose gel, the isotropic molecular conformation quickly orients in the field direction, reaching first a maximum overshoot'' orientation before it relaxes towards a somewhat less oriented but still anisotropic steady-state conformation. We present here a simple analytical model of this overshoot effect together with numerical results from a computer simulation of gel electrophoresis. The predicted dependence of the overshoot time and orientation upon field intensity and molecular size are in good agreement with experimental results. The dynamics of the overshoot involves U-shape conformations that disappear only after the internal elastic forces completely dominate the electric forces. It is also predicted that a different overshoot regime takes place for low electric fields and small molecular sizes, and that a primary and a secondary overshoot may appear for very large molecules.

  19. Assaying cooperativity of protein-DNA interactions using agarose gel electrophoresis.

    PubMed

    Williams, Tanya L; Levy, Daniel L

    2013-01-01

    DNA-binding proteins play essential roles in many cellular processes. Understanding on a molecular level how these proteins interact with their cognate sequences can provide important functional insights. Here, we describe a band shift assay in agarose gel to assess the mode of protein binding to a DNA molecule containing multiple protein-binding sites. The basis for the assay is that protein-DNA complexes display retarded gel electrophoresis mobility, due to their increased molecular weight relative to free DNA. The degree of retardation is higher with increasing numbers of bound protein molecules, thereby allowing resolution of complexes with differing protein-DNA stoichiometries. The DNA is radiolabeled to allow for visualization of both unbound DNA and all the different DNA-protein complexes. We present a quantitative analysis to determine whether protein binding to multiple sites within the same DNA molecule is independent or cooperative.

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

    PubMed

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

    PubMed

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

    2008-08-15

    The toxicity and inefficacy of actual organic drugs against Leishmaniosis justify research projects to find new molecular targets in Leishmania species including Leishmania infantum (L. infantum) and Leishmaniamajor (L. major), both important pathogens. In this sense, quantitative structure-activity relationship (QSAR) methods, which are very useful in Bioorganic and Medicinal Chemistry to discover small-sized drugs, may help to identify not only new drugs but also new drug targets, if we apply them to proteins. Dyneins are important proteins of these parasites governing fundamental processes such as cilia and flagella motion, nuclear migration, organization of the mitotic splinde, and chromosome separation during mitosis. However, despite the interest for them as potential drug targets, so far there has been no report whatsoever on dyneins with QSAR techniques. To the best of our knowledge, we report here the first QSAR for dynein proteins. We used as input the Spectral Moments of a Markov matrix associated to the HP-Lattice Network of the protein sequence. The data contain 411 protein sequences of different species selected by ClustalX to develop a QSAR that correctly discriminates on average between 92.75% and 92.51% of dyneins and other proteins in four different train and cross-validation datasets. We also report a combined experimental and theoretic study of a new dynein sequence in order to illustrate the utility of the model to search for potential drug targets with a practical example. First, we carried out a 2D-electrophoresis analysis of L. infantum biological samples. Next, we excised from 2D-E gels one spot of interest belonging to an unknown protein or protein fragment in the region M<20,200 and pI<4. We used MASCOT search engine to find proteins in the L. major data base with the highest similarity score to the MS of the protein isolated from L. infantum. We used the QSAR model to predict the new sequence as dynein with probability of 99.99% without

  2. Studies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis

    SciTech Connect

    Takai, N.A.; Filetti, S.; Rapoport, B.

    1981-01-01

    Highly purified bovine TSH (stored in solution at -70 C) was radioiodinated by the stoichiometric chloroamine-T method. The iodinated material ws subjected to analytical polyacrylamide disc gel electrophoresis. TSH was eluted from gel slices (1 mm width) and was analyzed for radioactivity and bioactivity. The latter was determined using the cultured thyroid cell cAMP response assay. Radioactivity in the TSH preparation migrated separately from bioactivity, but concordant with the protein bands observed in gels run in parallel. Further studies performed on bovine TSH purified in our laboratory, as well as on a different TSH preparation of exceptionally high potency (both stored as lyophilized powder) revealed a different pattern, with TSH bioactivity and radioactivity eluting concurrently. Iodination of TSH did not alter its electrophoretic migration on disc gel electrophoresis. In all preparations polymorphism of TSH bioactivity was observed, with at least four separate protein bands containing TSH bioactivity being present in our preparation. The relationship between the degree of iodination and retention of TSH bioactivity was examined. Incorporation of /sup 125/I into TSH was greatly different at two different concentrations of chloramine-T. Despite this, however, the progressive loss of TSH bioactivity was similar at both concentrations, indicating that incorporation of iodine into the TSH molecule is not itself responsible for the decrease in bioactivity. These studies indicate variability among different TSH preparations in terms of their retention of bioactivity. Significant loss of TSH bioactivity appears to occur during storage in solution. The damage to the biological activity of TSH during the iodination procedure is more likely related to the oxidation process than to the incorporation of iodine.

  3. Simultaneous detection of single nucleotide polymorphisms and copy number variations in the CYP2D6 gene by multiplex polymerase chain reaction combined with capillary electrophoresis.

    PubMed

    Liao, Hsiao-Wei; Tsai, I-Lin; Chen, Guan-Yuan; Kuo, Chun-Ting; Wei, Ming-Feng; Hwang, Tzung-Jeng; Chen, Wei J; Shen, Li-Jiuan; Kuo, Ching-Hua

    2013-02-06

    CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes.

  4. A new method for 2D gel spot alignment: application to the analysis of large sample sets in clinical proteomics

    PubMed Central

    Pérès, Sabine; Molina, Laurence; Salvetat, Nicolas; Granier, Claude; Molina, Franck

    2008-01-01

    Background In current comparative proteomics studies, the large number of images generated by 2D gels is currently compared using spot matching algorithms. Unfortunately, differences in gel migration and sample variability make efficient spot alignment very difficult to obtain, and, as consequence most of the software alignments return noisy gel matching which needs to be manually adjusted by the user. Results We present Sili2DGel an algorithm for automatic spot alignment that uses data from recursive gel matching and returns meaningful Spot Alignment Positions (SAP) for a given set of gels. In the algorithm, the data are represented by a graph and SAP by specific subgraphs. The results are returned under various forms (clickable synthetic gel, text file, etc.). We have applied Sili2DGel to study the variability of the urinary proteome from 20 healthy subjects. Conclusion Sili2DGel performs noiseless automatic spot alignment for variability studies (as well as classical differential expression studies) of biological samples. It is very useful for typical clinical proteomic studies with large number of experiments. PMID:18957120

  5. Two-Dimensional Gel Electrophoresis of DNA Replication Intermediates in Schizosaccharomyces pombe.

    PubMed

    Sánchez, Mar; Antequera, Francisco

    2017-07-21

    Two-dimensional gel electrophoresis allows direct detection of DNA replication and recombination intermediates in preparations of total genomic DNA. This technique is widely used to identify replication origins in the yeast genome and is based on the different mobility in agarose gels of linear and branched DNA molecules depending on their mass and structure. During the first dimension, low-voltage and a low-percentage agarose gel favors separation of the molecules by their mass and minimizes the effect of their structure. In contrast, during the second dimension, a higher voltage, a higher percentage agarose gel, and the presence of ethidium bromide significantly delays the migration of branched structures relative to linear molecules of the same mass. This technique is appropriate for the detection of replication initiation and, therefore, an active origin of replication, within regions of approximately 2.5-5.5 kb. Consequently, it is not well suited for genome-wide replication analyses. © 2018 Cold Spring Harbor Laboratory Press.

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  7. A model for the separation of large DNA molecules by crossed field gel electrophoresis.

    PubMed

    Southern, E M; Anand, R; Brown, W R; Fletcher, D S

    1987-08-11

    The idea that large DNA molecules adopt a stretched conformation as they pass through gels suggests a simple mechanism for the separation of DNA by crossed field electrophoresis: at each change in field direction a DNA molecule takes off in the new direction of the field by a movement which is led by what was formerly its back end. The effect of this ratcheting motion is to subtract from the DNA molecule's forward movement, at each step, an amount which is proportional to its length. We find that this model explains most of the features of the separation, and we describe experiments, using a novel electrophoresis apparatus, which support the model. The apparatus turns the gel between two preset orientations in a uniform electric field at preset time intervals. This separation method has the practical advantage over some others that the DNA molecules follow straight tracks. A further advantage is that the parameters which determine the separation are readily predicted from the simple theory describing their motion.

  8. A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

    PubMed Central

    Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

    2012-01-01

    In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on. PMID:22272223

  9. Investigating DNA Migration in Pulsed Fields Using a Miniaturized Field Inversion Gel Electrophoresis System

    PubMed Central

    Chen, Xiaojia; Ugaz, Victor M.

    2010-01-01

    Pulsed field gel electrophoresis (PFGE) is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here we present a simple miniaturized field inversion gel electrophoresis (FIGE) apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 hours using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N−0.5 over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively. PMID:19053074

  10. Intra-species chromosome-length polymorphism in Geotrichum candidum revealed by pulsed field gel electrophoresis.

    PubMed

    Gente, Stéphanie; Desmasures, Nathalie; Jacopin, Cyril; Plessis, Ghislaine; Beliard, Martine; Panoff, Jean-Michel; Guéguen, Micheline

    2002-06-05

    Geotrichum candidum is an ascomycetous anamorph yeast-like fungus found in various habitats. It is a component of the natural flora of milk and is used as a maturing agent for both soft and hard cheeses. This microorganism displays phenotypic variability and may act as an opportunist pathogen, causing geotrichosis. Cytological analysis of G. candidum strain ATCC 204307 showed this strain to have eight chromosomes. We prepared chromosomal DNA from 13 strains of G. candidum differing in habitat and morphotype. We used pulsed field gel electrophoresis (PFGE) in two sets of conditions to determine the size of the chromosomal DNA molecules. The strains investigated had five to eight chromosomes, 0.6 to 4.5 Mb in size. We estimated genome size in these 13 strains to be between 11 and 19 Mb. Pulsed-field gel electrophoresis profiles showed a high degree of polymorphism, indicating considerable variability between strains. Genome size and the presence of large chromosomes appeared to be correlated with morphotype. Strains with a mold-like or intermediate morphotype tended to have larger genomes than strains with a yeast-like morphotype did.

  11. Poultry digestive microflora biodiversity as indicated by denaturing gradient gel electrophoresis.

    PubMed

    Hume, M E; Kubena, L F; Edrington, T S; Donskey, C J; Moore, R W; Ricke, S C; Nisbet, D J

    2003-07-01

    Populations of digestive microflora in chickens change with age and are affected by diet, stressors, and performance enhancers. Culturing techniques used to profile a bacterial community inadvertently select for some organisms while excluding others. Several molecular-based techniques have been used to profile mixed microbial populations on the basis of DNA extracted from the entire community. Denaturing gradient gel electrophoresis was used in the present study to examine PCR-amplified fragments (amplicons) of a 16S ribosomal DNA variable region from predominant digestive bacteria. The objective of the study was to examine changes in digestive microbial communities of developing Leghorn chicks and molted Leghorn hens. Dendrograms of amplicon patterns indicated approximately 51% similarity between cecal bacteria composition in Leghorn chicks less than 20 d old and chicks greater than 20 d old. Cecal communities in Leghorn chicks given a competitive exclusion culture exhibited 21% correlation at all ages with those in control chicks. Nonmolted and molted hens had 40% similarity between cecal communities, whereas diets with low calcium (0.8% wt/wt) and excess zinc (2,800 mg/kg) lessened population differences (90% similarity). Results indicated the potential usefulness of the molecular-based denaturing gradient gel electrophoresis to monitor changes in digestive bacterial communities in chickens.

  12. A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics.

    PubMed

    Guo, Chen-Gang; Shang, Zhi; Yan, Jian; Li, Si; Li, Guo-Qing; Liu, Rong-Zhong; Qing, Ying; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-05-01

    Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins.

  13. Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products.

    PubMed

    Jimenez, Maria S; Luque-Alled, Jose M; Gomez, Teresa; Castillo, Juan R

    2016-05-01

    Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE).

  14. Agarose-gel electrophoresis for the quality assurance and purity of heparin formulations.

    PubMed

    Volpi, Nicola; Buzzega, Dania

    2012-01-01

    The adulteration of raw heparin (Hep) with a synthetic oversulfated chondroitin sulfate (OSCS) not found in nature produced in 2007-2008 a global crisis giving rise to the development of additional, new and specific methods for its quality assurance and purity. In this study, a simple and sensitive agarose-gel electrophoresis method has been developed for the visualization of OSCS in Hep samples along with other natural glycosaminoglycans possibly present as "process-related impurities", in particular dermatan sulfate (DS) and chondroitin sulfate (CS). Agarose-gel electrophoresis under non-conventional conditions is able to separate OSCS from Hep with its two components, the slow-moving and fast-moving species, DS and CS by performing separation for 15 h (overnight) and under high voltage (100 mA, ∼200 V). Densitometric scanning enabled us to calculate a limit of detection of ∼0.5 μg OSCS with a linear behaviour from 0.1 to 5 μg, comparable to CS/DS. Contaminated samples from Hep manufacturers were analyzed and quantitative data were found comparable to previous studies. Due to its capacity to process many samples in a single run and to the equipment commonly available in laboratories, this analytical method would be suitable for the identification and quantification of contamination by other polysaccharides, in particular OSCS and DS, within Hep preparations and formulations.

  15. Use of pulsed-field gel electrophoresis to measure DNA damage and repair

    SciTech Connect

    Scicchitano, D.A. New York Univ., New York )

    1991-03-11

    A method is described here for the analysis of single-strand break formation and repair in genomic DNA. The procedure involves exposing cells to a DNA-damaging agent, allowing time for recovery, and embedding the cells in agarose. After lysis and digestion with a protease, the DNA, which remains in the agarose plug, is denatured with glyoxal and separated by pulsed-field gel electrophoresis. The DNA in the gel is then transferred to a support membrane and quantitated with a radioanalytic imaging system to determine the average size of the DNA at each time point of recovery. The results indicate that the repair of methyl-induced breaks in total genomic DNA is approximately 80% complete in 48 hr in CHO B11 and ARL 14 cells exposed to dimethyl sulfate. These results are in agreement with those obtained by using other techniques like alkaline sucrose sedimentation. The method developed and described here has several advantages over existing techniques for repair measurements: It can be used to monitor genotoxic agents that nick DNA, to study the removal of breaks from genomic DNA, and to test for repair of damage in specific domains of chromatin that would be too large to examine by conventional electrophoresis.

  16. A control method to inspect the compositional authenticity of Minas Frescal cheese by gel electrophoresis.

    PubMed

    Magenis, Renata B; Prudêncio, Elane S; Molognoni, Luciano; Daguer, Heitor

    2014-08-20

    This study introduces a qualitative method to inspect the compositional authenticity of white nonripened cheeses like Minas Frescal, a typical Brazilian cheese, especially when irregular replacement of milk by whey is suspected. A sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) method, followed by image densitometry, was validated. Cheeses were freeze-dried to electrophoresis, and β-lactoglobulin (β-LG) was chosen as the adulteration marker. In gel trypsin digestion followed by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry provided its identification. Cheeses with a minimum of 14 mg·g(-1) of β-LG are considered to be adulterated. The method shows satisfactory precision with a detection limit of 7 mg·g(-1). Forty-two commercial samples from inspected establishments were then assessed and subjected to cluster analysis. Compliant and noncompliant groups were set with 24 (57%) authentic samples and 18 (43%) adulterated samples, respectively, showing that proper analytical monitoring is required to inhibit this practice.

  17. An improved method for Southern DNA and Northern RNA blotting using a Mupid-2 Mini-Gel electrophoresis unit.

    PubMed

    Furuya, Hirokazu; Yamada, Takeshi; Ikezoe, Koji; Ohyagi, Yasumasa; Fukumaki, Yasuyuki; Fujii, Naoki

    2006-08-31

    An improved method for Southern DNA and Northern RNA blotting using the Mupid-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.

  18. Analysis of Soluble Proteins in Natural Cordyceps sinensis from Different Producing Areas by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Two-dimensional Electrophoresis.

    PubMed

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

    2017-01-01

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

  19. Analysis of Soluble Proteins in Natural Cordyceps sinensis from Different Producing Areas by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Two-dimensional Electrophoresis

    PubMed Central

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

    2017-01-01

    Background: As one of the bioactive components in Cordyceps sinensis (CS), proteins were rarely used as index components to study the correlation between the protein components and producing areas of natural CS. Objective: Protein components of 26 natural CS samples produced in Qinghai, Tibet, and Sichuan provinces were analyzed and compared to investigate the relationship among 26 different producing areas. Materials and Methods: Proteins from 26 different producing areas were extracted by Tris-HCl buffer with Triton X-100, and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Results: The SDS-PAGE results indicated that the number of protein bands and optical density curves of proteins in 26 CS samples was a bit different. However, the 2-DE results showed that the numbers and abundance of protein spots in protein profiles of 26 samples were obviously different and showed certain association with producing areas. Conclusions: Based on the expression values of matched protein spots, 26 batches of CS samples can be divided into two main categories (Tibet and Qinghai) by hierarchical cluster analysis. SUMMARY The number of protein bands and optical density curves of proteins in 26 Cordyceps sinensis samples were a bit different on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profilesNumbers and abundance of protein spots in protein profiles of 26 samples were obvious different on two-dimensional electrophoresis mapsTwenty-six different producing areas of natural Cordyceps sinensis samples were divided into two main categories (Tibet and Qinghai) by Hierarchical cluster analysis based on the values of matched protein spots. Abbreviations Used: SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2-DE: Two-dimensional electrophoresis, Cordyceps sinensis: CS, TCMs: Traditional Chinese medicines PMID:28250651

  20. The impact of two-dimensional pulsed-field gel electrophoresis techniques for the consistent and complete mapping of bacterial genomes: refined physical map of Pseudomonas aeruginosa PAO.

    PubMed Central

    Römling, U; Tümmler, B

    1991-01-01

    The SpeI/DpnI map of the 5.9 Mb Pseudomonas aeruginosa PAO (DSM 1707) genome was refined by two-dimensional (2D) pulsed-field gel electrophoresis techniques (PFGE) which allow the complete and consistent physical mapping of any bacterial genome of interest. Single restriction digests were repetitively separated by PFGE employing different pulse times and ramps in order to detect all bands with optimum resolution. Fragment order was evaluated from the pattern of 2D PFGE gels: 1. Partial-complete digestion. A partial restriction digest was separated in the first dimension, redigested to completion, and subsequently perpendicularly resolved in the second dimension. 2D-gel comparisons of the ethidium bromide stain of all fragments and of the autoradiogram of end-labeled partial digestion fragments was nearly sufficient for the construction of the macrorestriction map. 2. Reciprocal gels. A complete restriction digest with enzyme A was run in the first dimension, redigested with enzyme B, and separated in the second orthogonal direction. The order of restriction digests was reverse on the second gel. In case of two rare-cutters, fragments were visualized by ethidium bromide staining or hybridization with genomic DNA. If a frequent and a rare cutter were employed, linking fragments were identified by end-labeling of the first digest. 3. A few small fragments were isolated by preparative PFGE and used as a probe for Southern analysis.--38 SpeI and 15 DpnI fragments were positioned on the map. The zero point was relocated to the 'origin of replication'. The anonymous mapping techniques described herein are unbiased by repetitive DNA, unclonable genomic regions, unfavourable location of restriction sites, or cloning artifacts as frequently encountered in other top-down or bottom-up approaches. Images PMID:1905802

  1. A comparative study of preparations of ovine lutenizing hormone by bioassay, immunodoublediffusion, radioimmunoassay, radioreceptor assay and polyacrylamide gel electrophoresis.

    PubMed

    Schlamowitz, M; Cronquist, J; Esfahani, M; Ward, D N

    1976-02-01

    Three preparations of ovine LH were compared for biological potency and by several in vitro parameters. All were found to be heterogenous by immunodoublediffusion and by electrophoresis in polyacrylamide gels. They all also showed similarities and/or differences with respect to their characteristics in immunodoublediffusion, radioimmunoassay, radioreceptor assay, gel electrophoresis and in dye-binding capacity, but in ways that preclude establishing a meaningful correlation between biopotency and the in vitro parameters or even among the in vitro parameters themselves. The implications of these findings for the use of these in vitro parameters for screening and assessing biological potencies of LH preparations and for inferring chemical and/or structural similarities between LH preparations are discussed. Aspects of polymorphism of LH, observed by electrophoresis on polyacrylamide gels, are also discussed.

  2. Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis.

    PubMed

    Lee, Kibeom; Pi, Kyungbae; Lee, Keeman

    2009-01-01

    A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea-urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.

  3. Proteomic comparisons of opaque and transparent variants of Streptococcus pneumoniae by two dimensional-differential gel electrophoresis.

    PubMed

    Chai, Melissa H; Weiland, Florian; Harvey, Richard M; Hoffmann, Peter; Ogunniyi, Abiodun D; Paton, James C

    2017-05-26

    Streptococcus pneumoniae (the pneumococcus) is a human pathogen, accounting for massive global morbidity and mortality. Although asymptomatic colonization of the nasopharynx almost invariably precedes disease, the critical determinants enabling pneumococcal progression from this niche to cause invasive disease are poorly understood. One mechanism proposed to be central to this transition involves opacity phase variation, whereby pneumococci harvested from the nasopharynx are typically transparent, while those simultaneously harvested from the blood are opaque. Here, we used two dimensional-differential gel electrophoresis (2D-DIGE) to compare protein expression profiles of transparent and opaque variants of 3 pneumococcal strains, D39 (serotype 2), WCH43 (serotype 4) and WCH16 (serotype 6A) in vitro. One spot comprising a mixture of capsular polysaccharide biosynthesis protein and other proteins was significantly up-regulated in the opaque phenotype in all 3 strains; other proteins were differentially regulated in a strain-specific manner. We conclude that pneumococcal phase variation is a complex and multifactorial process leading to strain-specific pathogenicity.

  4. Assessing CMT cell line stability by two dimensional polyacrylamide gel electrophoresis and mass spectrometry based proteome analysis.

    PubMed

    Zhang, Kelan; Wrzesinski, Krzysztof; Fey, Stephen J; Mose Larsen, Peter; Zhang, Xumin; Roepstorff, Peter

    2008-07-21

    Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high-, middle- or low-metastatic potential have been characterized in vivo. In this study, the comprehensive protein expression profiles of the CMT cell lines were analyzed at passages 5, 15 and 35 in order to assess the cell line stability. During the passages 5 to 15, the expression profiles of CMT cells remained reasonably stable as evidenced by only 0.7%, 3.9% and 1.1% proteins changed in CMT167(H), CMT64(M) and CMT170(L) respectively. However, the number of differentially expressed proteins were considerably increased at passage 35 in CMT64(M) and CMT170(L) while CMT167(H) remained stable. Based on our selection criteria, 22, 109 and 84 spots in CMT167(H), CMT64(M) and CMT170(L) were selected for protein identification by MS and 99 unique proteins were identified. Bioinformatics analysis indicated that most of these proteins participate in cellular metabolism. In conclusion, proteomics was found to be a useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs.

  5. Identification of 2D-gel proteins : a comparison of MALDI/TOF peptide mass mapping to {mu} LC-ESI tandem mass spectrometry.

    SciTech Connect

    Lim, H.; Hays, L. G.; Eng, J.; Tollaksen, S. L.; Giometti, C. S.; Holden, J. F.; Adams, M. W. W.; Reich, C. I.; Olsen, G. J.; Yates, J. R.; Biosciences Division; The Scripps Research Inst.; Univ. of Georgia; Univ. of Illinois

    2003-09-01

    A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

  6. High-resolution gel electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs.

    PubMed

    Giori, Luca; Tricomi, Flavia Marcella; Zatelli, Andrea; Roura, Xavier; Paltrinieri, Saverio

    2011-07-01

    The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: <0.20; 32/87), borderline proteinuric (BP; UPC ratio: 0.21-0.50; 15/87), or proteinuric (P; UPC ratio: >0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (κ = 0.59; κ = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/α(1)-globulin ratio (alb/α(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of α(1)-globulin >40.8% and an alb/α(1) ratio <0.84 can identify samples classified by SDS-AGE as affected by tubular proteinuria. In conclusion, both SDS-AGE and HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and α(1)-globulin or the alb/α(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria.

  7. Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

    PubMed Central

    Tang, Shuiquan; Chan, Winnie W. M.; Fletcher, Kelly E.; Seifert, Jana; Liang, Xiaoming; Löffler, Frank E.; Adrian, Lorenz

    2013-01-01

    Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family. PMID:23204411

  8. Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles.

    PubMed

    Zhao, Wenting; Yao, Chunlei; Luo, Xiaoteng; Lin, Li; Hsing, I-Ming

    2012-04-01

    We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.

  9. Automatic analysis of 2D polyacrylamide gels in the diagnosis of DNA polymorphisms.

    PubMed

    Koprowski, Robert; Wróbel, Zygmunt; Korzyńska, Anna; Chwiałkowska, Karolina; Kwaśniewski, Mirosław

    2013-07-08

    The analysis of polyacrylamide gels is currently carried out manually or automatically. In the automatic method, there are limitations related to the acceptable degree of distortion of lane and band continuity. The available software cannot deal satisfactorily with this type of situations. Therefore, the paper presents an original image analysis method devoid of the aforementioned drawbacks. This paper examines polyacrylamide gel images from Li-Cor DNA Sequencer 4300S resulting from the use of the electrophoretic separation of DNA fragments. The acquired images have a resolution dependent on the length of the analysed DNA fragments and typically it is MG×NG=3806×1027 pixels. The images are saved in TIFF format with a grayscale resolution of 16 bits/pixel. The presented image analysis method was performed on gel images resulting from the analysis of DNA methylome profiling in plants exposed to drought stress, carried out with the MSAP (Methylation Sensitive Amplification Polymorphism) technique. The results of DNA polymorphism analysis were obtained in less than one second for the Intel Core™ 2 Quad CPU Q9300@2.5GHz, 8GB RAM. In comparison with other known methods, specificity was 0.95, sensitivity = 0.94 and AUC (Area Under Curve) = 0.98. It is possible to carry out this method of DNA polymorphism analysis on distorted images of polyacrylamide gels. The method is fully automatic and does not require any operator intervention. Compared with other methods, it produces the best results and the resulting image is easy to interpret. The presented method of measurement is used in the practical analysis of polyacrylamide gels in the Department of Genetics at the University of Silesia in Katowice, Poland.

  10. Automatic analysis of 2D polyacrylamide gels in the diagnosis of DNA polymorphisms

    PubMed Central

    2013-01-01

    Introduction The analysis of polyacrylamide gels is currently carried out manually or automatically. In the automatic method, there are limitations related to the acceptable degree of distortion of lane and band continuity. The available software cannot deal satisfactorily with this type of situations. Therefore, the paper presents an original image analysis method devoid of the aforementioned drawbacks. Material This paper examines polyacrylamide gel images from Li-Cor DNA Sequencer 4300S resulting from the use of the electrophoretic separation of DNA fragments. The acquired images have a resolution dependent on the length of the analysed DNA fragments and typically it is MG×NG=3806×1027 pixels. The images are saved in TIFF format with a grayscale resolution of 16 bits/pixel. The presented image analysis method was performed on gel images resulting from the analysis of DNA methylome profiling in plants exposed to drought stress, carried out with the MSAP (Methylation Sensitive Amplification Polymorphism) technique. Results The results of DNA polymorphism analysis were obtained in less than one second for the Intel Core™ 2 Quad CPU Q9300@2.5GHz, 8GB RAM. In comparison with other known methods, specificity was 0.95, sensitivity = 0.94 and AUC (Area Under Curve) = 0.98. Conclusions It is possible to carry out this method of DNA polymorphism analysis on distorted images of polyacrylamide gels. The method is fully automatic and does not require any operator intervention. Compared with other methods, it produces the best results and the resulting image is easy to interpret. The presented method of measurement is used in the practical analysis of polyacrylamide gels in the Department of Genetics at the University of Silesia in Katowice, Poland. PMID:23835039

  11. Determination of DNA methylation by COBRA: a comparative study of CGE with LIF detection and conventional gel electrophoresis.

    PubMed

    Goedecke, Simon; Schlosser, Sabrina; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

    2009-09-01

    DNA methylation as an epigenetic modification of the human genome is under emphatic investigation. Several studies have demonstrated a role of DNA methylation in oncogenesis. In conjunction with histone modifications, DNA methylation may cause the formation of heterochromatin and thus mediate the inactivation of gene transcription. It is important to develop methods that allow for an accurate quantification of the amount of DNA methylation in particular DNA regions, to gain information concerning the threshold of methylation levels necessary for gene inactivation. In this article, a CGE method with on-column LIF detection using SYBR Green is compared with a conventional slab-gel electrophoresis. We thus investigate the validity to analyze DNA methylation in the samples of a combined bisulfite restriction analysis. It is demonstrated that CGE is superior to gel electrophoresis in means of linearity, precision, accuracy, automatization (high throughput), and sample consumption. However, gel electrophoresis is easier to perform (simple devices, no PC usage), and the running costs are comparatively low. A further advantage of CGE is the sparse use of toxic compounds (MeOH and SYBR Green), whereas gel electrophoresis is performed in polyacrylamide gels with ethidium bromide staining.

  12. Characterization of nosocomial Serratia marcescens isolates: comparison of Fourier-transform infrared spectroscopy with pulsed-field gel electrophoresis of genomic DNA fragments and multilocus enzyme electrophoresis.

    PubMed

    Irmscher, H M; Fischer, R; Beer, W; Seltmann, G

    1999-07-01

    A total of 66 Serratia marcescens isolates from 46 patients was investigated by macrorestriction using XbaI followed by pulsed-field gel electrophoresis. 7 restriction fragment patterns attributable to more than one patient and 9 individual patterns were identified. The isolates were additionally characterized by multilocus enzyme electrophoresis and Fourier-transform infrared spectroscopy. The macrorestriction patterns and the multilocus enzyme electrophoresis patterns corresponded fairly well while the classifications derived from these methods were not completely congruent. The grouping achieved by Fourier-transform infrared spectroscopy on the basis of high (> 1000) and moderately high heterogeneity values (300) was consistent with the macrorestriction results. Grouping on a lower heterogeneity level did not contribute to further discrimination. In general, Fourier-transform infrared spectroscopy was less discriminatory than the two other methods, but easier to perform. Therefore, laboratories equipped with the necessary devices may use it to rapidly select bacterial isolates for macrorestriction or other well established characterization procedures.

  13. Cu2+-assisted two dimensional charge-mass double focusing gel electrophoresis and mass spectrometric analysis of histone variants.

    PubMed

    Zhang, Wenyang; Tang, Xuemei; Ding, Mengjie; Zhong, Hongying

    2014-12-10

    Abundant isoforms and dynamic posttranslational modifications cause the separation and identification of histone variants to be experimentally challenging. To meet this need, we employ two-dimensional electrophoretic gel separation followed by mass spectrometric detection which takes advantage of the chelation of Cu(2+) with amino acid residues exposed on the surfaces of the histone proteins. Acid-extracted rat liver histones were first mixed with CuSO4 solution and then separated in one dimension with triton-acid-urea (TAU) gel electrophoresis and in a second dimension using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separations result from both the changes in charge and mass upon Cu(2+) chelation. Identities of each separated gel bands were obtained by using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). It was found that the migration of H3 histone isoforms of rat liver is markedly affected by the use of Cu(2+) ions.

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

    PubMed Central

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

    2017-01-01

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

  15. Pulsed-field gel electrophoresis analysis of multicellular DNA double-strand break damage and repair.

    PubMed

    Joshi, Nina; Grant, Stephen G

    2014-01-01

    This assay quantifies the extent of double-strand break (DSB) DNA damage in cell populations embedded in agarose and analyzed for migratory DNA using pulsed-field gel electrophoresis with ethidium bromide staining. The assay can measure preexisting damage as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells can be allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in the lanes in comparison to the total amount of DNA fragmentation following determination of an optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.

  16. Characterization of Listeria monocytogenes isolates from cattle and ground beef by pulsed-field gel electrophoresis.

    PubMed

    Foerster, Claudia; Vidal, Lorena; Troncoso, Miriam; Figueroa, Guillermo

    2012-01-01

    The aims of this study were to determine the occurrence of Listeria monocytogenes in cattle feces and ground beef, to characterize these strains by pulsed-field gel electrophoresis and to compare them to three listeria strains found in humans. Cattle from different origins (n = 250) and ground beef obtained from supermarkets (n = 40) were sampled. The results show low occurrence in cattle feces (0.4 %) but a higher presence in ground beef (37 %). An important part of the ground beef strains (80 %) had > 95 % similarity with a strain isolated from a human sporadic case and the ATCC 19115 used as control. The strain isolated from cattle feces had 93 % similarity to clone 009, previously associated with a listeriosis outbreak related to cheese. Cattle and ground beef can harbor virulent L. monocytogenes strains. Further studies in animals and animal products are needed to improve listeriosis control.

  17. Molecular Typing by Pulsed-Field Gel Electrophoresis of Spanish Animal and Human Listeria monocytogenes Isolates

    PubMed Central

    Vela, A. I.; Fernandez-Garayzabal, J. F.; Vazquez, J. A.; Latre, M. V.; Blanco, M. M.; Moreno, M. A.; de la Fuente, L.; Marco, J.; Franco, C.; Cepeda, A.; Rodriguez Moure, A. A.; Suarez, G.; Dominguez, L.

    2001-01-01

    A total of 153 strains of Listeria monocytogenes isolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains of L. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis. PMID:11722943

  18. Evaluation of Pulsed-Field Gel Electrophoresis Profiles for Identification of Salmonella Serotypes▿ †

    PubMed Central

    Zou, Wen; Lin, Wei-Jiun; Foley, Steven L.; Chen, Chun-Houh; Nayak, Rajesh; Chen, James J.

    2010-01-01

    Pulsed-field gel electrophoresis (PFGE) is a standard typing method for isolates from Salmonella outbreaks and epidemiological investigations. Eight hundred sixty-six Salmonella enterica isolates from eight serotypes, including Heidelberg (n = 323), Javiana (n = 200), Typhimurium (n = 163), Newport (n = 93), Enteritidis (n = 45), Dublin (n = 25), Pullorum (n = 9), and Choleraesuis (n = 8), were subjected to PFGE, and their profiles were analyzed by random forest classification and compared to conventional hierarchical cluster analysis to determine potential predictive relationships between PFGE banding patterns and particular serotypes. Cluster analysis displayed only the underlying similarities and relationships of the isolates from the eight serotypes. However, for serotype prediction of a nonserotyped Salmonella isolate from its PFGE pattern, random forest classification provided better accuracy than conventional cluster analysis. Discriminatory DNA band class markers were identified for distinguishing Salmonella serotype Heidelberg, Javiana, Typhimurium, and Newport isolates. PMID:20631109

  19. Molecular analysis of chromosomal rearrangements using pulsed field gel electrophoresis and somatic cell hybrids

    SciTech Connect

    Davis, L.M. )

    1991-01-01

    Many human genetic diseases, including some cancers, are characterized by consistent chromosome abnormalities, such as deletions and translocations. Analyses of these mutations often prove crucial to the eventual cloning and characterization of the gene(s) responsible for the disease. Two methods for analyzing these chromosome abnormalities have been developed in recent years: somatic cell hybridization and pulsed field gel electrophoresis (PFGE). Somatic cell hybridization is a technique for segregating an aberrant chromosome from its normal homologue in a cell derived from an unrelated species, which is usually a rodent. Demonstrations of these analytic techniques are presented, using as an example chromosomal abnormalities involving human chromosome band 11p13, the locus for the Wilms' tumor, aniridia, genitourinary abnormality, and mental retardation (WAGR) syndrome.

  20. Protein expression of sensory and motor nerves: Two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Ren, Zhiwu; Wang, Yu; Peng, Jiang; Zhang, Li; Xu, Wenjing; Liang, Xiangdang; Zhao, Qing; Lu, Shibi

    2012-02-15

    The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product (gi|55628), glyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylglutathione lyase, adenylate kinase isozyme 1, two unnamed proteins products (gi|55628 and gi|1334163), and poly(rC)-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

  1. An exactly solvable Ogston model of gel electrophoresis: X. Application to high-field separation techniques.

    PubMed

    Gauthier, Michel G; Slater, Gary W

    2003-01-01

    Recently, we generalized our lattice model of gel electrophoresis to study the net velocity of particles being pulled by a high-intensity electric field through an arbitrary distribution of immobile obstacles (Gauthier, M. G., Slater, G. W., J. Chem. Phys. 2002, 117, 6745-6756). In this article, we show how the high-field version of our model can be used to compare the velocity of particles with different electric charges and/or physical sizes. We then investigate specific two-dimensional distributions of obstacles that can be used to separate particles, e.g., in a microfluidic device. More precisely, we compare the velocity of differently charged or sized analytes in sieving, trapping and deflecting systems to model various electrophoretic separation techniques. In particular, we study the nonlinear effects present in ratchet systems and how they can be combined with time-asymmetric pulsed fields to provide new modes of separation.

  2. Non-denaturing gel electrophoresis system for the purification of membrane bound proteins

    SciTech Connect

    Cavinato, A.G.; Macleod, R.M.; Ahmed, M.S.

    1988-01-01

    A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an /sup 3/H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.

  3. Campylobacter coli pulsed field gel electrophoresis genotypic diversity among sows and piglets in a farrowing barn.

    PubMed

    Hume, Michael E; Droleskey, Robert E; Sheffield, Cynthia L; Harvey, Roger B

    2002-08-01

    Genotypes of Campylobacter coli isolates from feces of three sows and rectal swabs of 17 piglets were examined by pulsed field gel electrophoresis (PFGE). All of the animals originated from a single farrowing barn of a farrow-to-finish swine operation. Five Campylobacter colonies were picked from a single agar plate for each sample after broth enrichment and growth on Campy-Cefex agar. Genotypes were examined by PFGE after genomic DNA digestion with SmaI and SacII restriction endonucleases. Twenty SmaI genotypes and 12 SacII genotypes were detected among 99 Campylobacter coli isolates. There was no pattern of shared genotypes between sows and their respective piglets, nor between littermates. Results indicate that a high number of Campylobacter genotypes may coexist in related pigs from a single housing facility.

  4. Mapping of Escherichia Coli Chromosomal Tn5 and F Insertions by Pulsed Field Gel Electrophoresis

    PubMed Central

    Smith, C. L.; Kolodner, R. D.

    1988-01-01

    A low resolution Not I physical map of Escherichia coli was recently constructed. In this report we demonstrated that this map can be used to map Tn5 and F insertions physically. The transposon, Tn5, contains Not I recognition sequences in its IS50 sequences. F plasmid contains an unmapped Not I site. Hence, the location of Tn5 and F in the chromosome can be mapped by identifying the location of the introduced Not I sites using pulsed field gel electrophoresis. The physical mapping of genetically mapped Tn5 insertions confirm the previously constructed Not I map and helps align the E. coli physical and genetic maps. The use of Tn5 can assist the construction of both physical and genetic maps for microorganisms lacking such maps. Variations on this approach will facilitate physical mapping with a wide variety of organisms, enzymes, and genetic elements. PMID:2840334

  5. Microdisc gel electrophoresis in sodium dodecyl sulfate of organic material from rat otoconial complexes

    NASA Technical Reports Server (NTRS)

    Ross, M. D.; Pote, K. G.; Rarey, K. E.; Verma, L. M.

    1981-01-01

    The gravity receptors of all vertebrates utilize a 'test mass' consisting of a complex arrangement of mineral and organic substance that lies over the sensory receptor areas. In most vertebrates, the mineral is a polymorph of calcium carbonate in the form of minute, single crystals called otoconia. An investigation is conducted to determine the number of proteins in otoconial complexes and their molecular weights. The investigation makes use of a microdisk gel electrophoresis method reported by Gainer (1971). The most important finding of the reported research is that analysis of the proteins of the organic material of the otoconial complexes is possible when sensitive microanalytical methods are employed. Further modification of the basic technique employed and the inclusion of other sensitive staining methods should mean that, in the future, protein separation by molecular weight will be possible in sample pools containing only two otoconial masses.

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

    PubMed

    Holmberg, Mats; Nollen, Ellen A A

    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 expression of fluorescently tagged aggregation-prone proteins have been used to screen for genetic modifiers of aggregation. To establish the role of modifying factors in the generation of aggregation intermediates, a method has been developed using native agarose gel electrophoresis (NAGE) that enables parallel screening of aggregation patterns of fluorescently labeled aggregation-prone proteins. Together with microscopy-based genetic screens this method can be used to identify modifiers of protein aggregation and characterize their molecular function. Although described here for analyzing aggregates in C. elegans, NAGE can be adjusted for use in other model organisms as well as for cultured cells.

  7. Pulsed-field gel electrophoresis patterns of Escherichia coli O157 isolates from Kansas feedlots.

    PubMed

    Sargeant, J M; Shi, X; Sanderson, M W; Renter, D G; Nagaraja, T G

    2006-01-01

    This study investigated the prevalence and distribution of Escherichia coli O157 genetic types within and among feedlots using pulsed-field gel electrophoresis to separate XbaI-digested DNA. The study population consisted of 300 pens of cattle in 30 feedlots in Kansas that were sampled (feces, water, and water sediment) within a month of being shipped for slaughter. The prevalence of E. coli O157 was 8.5% in feces, 3.1% in water, and 4.5% in water sediment samples. A total of 424 E. coli O157 isolates were characterized by pulsed-field gel electrophoresis, and 139 subtypes (100% Dice similarity with no band differences) were identified. The majority of subtypes (70/139) was identified only once, but nine were identified 10 or more times. Identical subtypes were recovered from both feces and water tanks in 10 feedlots. The majority of subtypes were identified in only one feedlot, and the number of subtypes ranged from one to 23 within a feedlot and from one to seven within a pen. There were 10 feedlots with at least 15 positive samples. In these 10 feedlots, the most common subtype accounted for 16.9-78.6% of the isolates. Common subtypes differed among feedlots. In eight of the 10 feedlots, the most common subtype was identified in multiple pens. The results support a complex ecology for E. coli O157 in feedlot operations, with factors associated with exposure and transmission likely acting at a common level for multiple feedlots, within feedlots, and within pens of cattle.

  8. Conformational Entropy Mechanism for Periodic Motion of DNA under Constant-Field Gel Electrophoresis

    NASA Astrophysics Data System (ADS)

    Azuma, Ryuzo; Takayama, Hajime

    2006-06-01

    Entropic elasticity of a single charged polymer undergoing gel electrophoresis is a fundamental theme of polymer statistical physics since the discovery of “periodic” behavior in constant field gel electrophoresis (CFGE). In the present work we address the problem numerically by two steps. In the first step, we carry out Brownian dynamics (BD) simulations on CFGE by solving semi-microscopic Langevin equations of a polymer consisting of beads separated by a mean distance much smaller than the Kuhn length. Results are analyzed based on coarse-graining over the Kuhn length scale. We show the averaged elongation-contraction motion involves asymmetric V-shaped configurations whose shorter arm length depends on the field and the temperature consistently with what is expected when the BD chain is described by the freely-jointed chain (FJC) model with a suitable Kuhn length. To our knowledge, this is the first numerical confirmation of the FJC model itself from a submicroscopic description of polymer motion. The saturation of chain mobility in high fields agrees well with the nonlinear dependence of this shorter arm length on the field. In the second step, we discuss the periodic elongation-contraction motion of the coarse-grained chain by such a simplified model as a one-dimensional chain consisting of beads, elastic strings, and obstacles. The results from these two chain models indicate that the periodic elongation-contraction motion of DNA under CFGE is self-organized by a balance between the field force and the conformational entropic force.

  9. Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

    PubMed

    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2011-05-01

    New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

  10. Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting.

    PubMed

    Cheng, Tsai-Mu; Huang, Pei-Chen; Pan, Ju-Pin; Lin, Kuan-Yu; Mao, Simon J T

    2007-04-15

    Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.

  11. Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis.

    PubMed Central

    Snell, R G; Wilkins, R J

    1986-01-01

    Modifications have been made to standard pulse field gel electrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of electrophoresis to 35 degrees C. This enabled the largest Saccharomyces cerevisiae chromosome to be reproducibly resolved. More impressively, it enabled the DNA of Candida albicans to be clearly resolved into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albicans chromosomes, which was estimated to be at least 5-10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highest molecular weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albicans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes. Images PMID:3520483

  12. Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis of freshwater photosynthetic sulfur bacteria.

    PubMed

    Osuna, M Begoña; Casamayor, Emilio O

    2011-01-01

    Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Cisó and Lake Vilar, NE Spain) where planktonic photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial biomass. Several extraction, solubilization, and electrophoresis methods were tested to develop an optimal protocol for the best resolution of the SDS-PAGE. Protein composition from different water depths and at different times of the year was visualized within a molecular mass range between 100 and 15 kDa yielding up to 20 different protein bands. Protein banding patterns were reproducible and changed in time and with depth in agreement with changes in photosynthetic bacteria composition. When a taxonomically stable community was followed in time, differences were observed in the intensity but not in the composition of the SDS-PAGE banding pattern. Three environmental variables directly related to the activity of sulfur bacteria (light, oxygen, and sulfide concentrations) had a significant effect on protein banding patterns and explained 33% of the variance. Changes in natural protein profiles of the bacterial blooms agreed with changes in species composition and in the in situ metabolic state of the populations.

  13. Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis.

    PubMed

    Lefkowitz, Roy B; Schmid-Schönbein, Geert W; Heller, Michael J

    2010-07-01

    The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

  14. DNA damage by carbon nanotubes using the single cell gel electrophoresis technique.

    PubMed

    Zeni, Olga; Scarfì, Maria Rosaria

    2010-01-01

    The single-cell gel electrophoresis (SCGE) or comet assay is a simple and sensitive method for quantitatively measuring DNA breakage and repair in individual cells. It can be applied to proliferating and non-proliferating cells and cells of those tissues, which are the first contact sites for mutagenic/carcinogenic substances. In this technique, cells are embedded in agarose, lysed, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. Cells with increased DNA damage display increased DNA migration from the nucleus toward the anode, which resembles the shape of a comet. The migration is observed by fluorescence microscopy after staining with a fluorescent DNA-binding dye, and the intensity of the comet tail reflects the number of DNA breaks. The assay is performed in almost all eukaryotic cells and has applications in many fields, including genetic toxicology, biomonitoring, ecotoxicology, medical, and nutritional research. The assay is a very sensitive tool to investigate the effect of carbon nanotubes on DNA of human cells in vitro. This chapter describes a procedure to perform the comet assay, in its alkaline version, on cell cultures treated with carbon nanotubes.

  15. Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

    PubMed Central

    Churchward, Matthew A; Butt, R Hussain; Lang, John C; Hsu, Kimberly K; Coorssen, Jens R

    2005-01-01

    Background The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. Results After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. Conclusion This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis. PMID:15941475

  16. Separation and recovery of nucleic acids with improved biological activity by acid-degradable polyacrylamide gel electrophoresis.

    PubMed

    Kim, Yoon Kyung; Kwon, Young Jik

    2010-05-01

    One of the fundamental challenges in studying biomacromolecules (e.g. nucleic acids and proteins) and their complexes in a biological system is isolating them in their structurally and functionally intact forms. Electrophoresis offers convenient and efficient separation and analysis of biomacromolecules but recovery of separated biomacromolecules is a significant challenge. In this study, DNAs of various sizes were separated by electrophoresis in an acid-degradable polyacrylamide gel. Almost 100% of the nucleic acids were recovered after the identified gel bands were hydrolyzed under a mildly acidic condition and purified using anion exchange resin. Further concentration by centrifugal filtration and a second purification using ion exchange column chromatography yielded 44-84% of DNA. The second conventional (non-degradable) gel electrophoresis confirmed that the nucleic acids recovered from acid-degradable gel bands preserved their electrophoretic properties through acidic gel hydrolysis, purification, and concentration processes. The plasmid DNA recovered from acid-degradable gel transfected cells significantly more efficiently than the starting plasmid DNA (i.e. improved biological activity via acid-degradable PAGE). Separation of other types of nucleic acids such as small interfering RNA using this convenient and efficient technique was also demonstrated.

  17. Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.

    PubMed

    Lengyel, T; Guttman, A

    1999-08-20

    Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated.

  18. Effects of coffee bean aroma on the rat brain stressed by sleep deprivation: a selected transcript- and 2D gel-based proteome analysis.

    PubMed

    Seo, Han-Seok; Hirano, Misato; Shibato, Junko; Rakwal, Randeep; Hwang, In Kyeong; Masuo, Yoshinori

    2008-06-25

    The aim of this study was 2-fold: (i) to demonstrate influences of roasted coffee bean aroma on rat brain functions by using the transcriptomics and proteomics approaches and (ii) to evaluate the impact of roasted coffee bean aroma on stress induced by sleep deprivation. The aroma of the roasted coffee beans was administered to four groups of adult male Wistar rats: 1, control group; 2, 24 h sleep deprivation-induced stress group (the stress group); 3, coffee aroma-exposed group without stress (the coffee group); and 4, the stress with coffee aroma group (the stress with coffee group). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of some known genes responsive to aroma or stress was performed using total RNA from these four groups. A total of 17 selected genes of the coffee were differently expressed over the control. Additionally, the expression levels of 13 genes were different between the stress group and the stress with coffee group: Up-regulation was found for 11 genes, and down-regulation was seen for two genes in the stress with coffee group. We also looked to changes in protein profiles in these four samples using two-dimensional (2D) gel electrophoresis; 25 differently expressed gel spots were detected on 2D gels stained by silver nitrate. Out of these, a total of nine proteins were identified by mass spectrometry. Identified proteins belonged to five functional categories: antioxidant; protein fate; cell rescue, defense, and virulence; cellular communication/signal transduction mechanism; and energy metabolism. Among the differentially expressed genes and proteins between the stress and the stress with coffee group, NGFR, trkC, GIR, thiol-specific antioxidant protein, and heat shock 70 kDa protein 5 are known to have antioxidant or antistress functions. In conclusion, the roasted coffee bean aroma changes the mRNA and protein expression levels of the rat brain, providing for the first time clues to the potential antioxidant or stress

  19. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

    PubMed

    Georgakilas, Alexandros G; Holt, Stewart M; Hair, Jessica M; Loftin, Charles W

    2010-12-01

    The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

  20. Application of multiplex PCR, pulsed-field gel electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci

    USDA-ARS?s Scientific Manuscript database

    The objective of the study was to use band-based molecular methods including BOX-PCR (Polymerase Chain Reaction) and Pulsed-Field Gel Electrophoresis (PFGE) to determine if genetically related enterococci were found among different stores, food types, or years. Enterococci were also characterized f...

  1. Genetic diversity demonstrated by pulsed field gel electrophoresis of Salmonella enterica isolates obtained from diverse sources in Mexico

    USDA-ARS?s Scientific Manuscript database

    This study was conducted to determine the genetic diversity of Salmonella isolates recovered from a variety of sources using pulsed-field gel electrophoresis (PFGE) to assess their possible relatedness. Salmonella was isolated from ca. 52% of samples from a pepper var. Bell production system. A to...

  2. Electrophoresis of /sup 35/S-labeled proteoglycans of polyacrylamide-agarose composite gels and their visualization by fluorography

    SciTech Connect

    Carney, S.L.; Bayliss, M.T.; Collier, J.M.; Muir, H.

    1986-01-01

    Techniques for the electrophoresis of /sup 35/S-labeled proteoglycans on polyacrylamide-agarose gel slabs and subsequent fixation, impregnation, and fluorography of such electrophoretograms have been developed. The procedure permits the examination of newly synthesized proteoglycan subspecies using a rapid technique, previously unavailable for these labeled molecules.

  3. Serum protein electrophoresis by using high-resolution agarose gel in clinically healthy and Aspergillus species-infected falcons.

    PubMed

    Kummrow, Maya; Silvanose, Christudas; Di Somma, Antonio; Bailey, Thomas A; Vorbrüggen, Susanne

    2012-12-01

    Serum protein electrophoresis has gained importance in avian medicine during the past decade. Interpretation of electrophoretic patterns should be based on species-specific reference intervals and the electrophoresis gel system. In this study, serum protein electrophoresis by using high-resolution agarose gels was performed on blood samples collected from 105 falcons, including peregrine falcons (Falco peregrinus), gyrfalcons (Falco rusticolus), saker falcons (Falco cherrug), red-naped shaheens (Falco pelegrinoides babylonicus), and hybrid falcons, that were submitted to the Dubai Falcon Hospital (Dubai, United Arab Emirates) between 2003 and 2006. Reference values were established in clinically healthy birds and compared with values from falcons infected with Aspergillus species (n = 32). Falcons with confirmed aspergillosis showed significantly lower prealbumin values, which is a novel finding. Prealbumin has been documented in many avian species, but further investigation is required to illuminate the diagnostic significance of this negative acute-phase protein.

  4. Immunoreactivity and two-dimensional gel-electrophoresis characterization of Egyptian cobra venom proteome.

    PubMed

    Almehdar, Hussein Abduelrahman; Adel-Sadek, Mahmoud Abass; Redwan, Elrashdy Moustafa

    2015-01-01

    The first and second (two) dimensional gel electrophoresis has a broad protein resolution power. It was used to separate and identify cobra venom proteome. The importance of characterizing venom proteins contents from the Egyptian elapidae, specifically neurotoxins, is based on the need to produce effective anti-venom. About 30-55distinct protein spots were identified on silver stained two-dimensional gels. Around two-thirds of the venom proteins displayed low a molecular weight and a migration into hydrophobic side. The venoms from Naja haja and Naja nigricollus showed 45-55 spots, while Walternnesia aegyptia had less (31-37) spots. The commercial prepared polyclonal antivenom had a strong signal for anionic and cationic venom protein spots with molecular weight 20-115 kDa. However, it showed weak or non immunoreactivity toward anionic low molecular weight spots (2.5-15 kDa). These results suggest the need to change the immunization schedule to include low molecular weight toxin-proteomes as separate dose or sequester injection.

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

    PubMed

    Singh, Sanjay Kumar; Ramaiah, Nagappa

    2011-05-01

    Denaturing gradient gel electrophoresis (DGGE) was used to elucidate spatial and temporal variations in bacterial community composition (BCC) from four locations along the central west coast of India. DNA extracts from 36 water samples collected from surface, mid-depth (-10 m) and dose to bottom (-20 m) during premonsoon, postmonsoon, monsoon were analyzed by PCRfor amplifying variable region of 16S rRNAgene and subsequently through DGGE. Prominent bands were excised, cloned and sequenced indicated the preponderance of gammaproteobacteria, bacteroidetes and cyanobacteria. Non-metric dimensional scaling of the DGGE gels indicated that the spatial variations in BCC were prominent among the sampling locations. Temporal variations in the BCC appear to be influenced by monsoonal processes. The canonical correspondence analyses suggest that the concentration of chlorophyll a and nitrate are two important environmental factors for both spatial and temporal variations in BCC. Chlorophyll a seems to be impart a top-down control of BCC while nitrate, the bottom-up control. Our results also suggest that BCC can vary over a small geographic distance in highly dynamic, seasonally predisposed tropical coastal waters.

  6. Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography.

    PubMed

    Xia, Zhi-Jun; Liu, Zhen; Kong, Fan-Zhi; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2017-08-12

    Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad-spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free-flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS-PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30-fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2-fold dilution but the latter had ∼13-fold dilution. Furthermore, Tricine-SDS-PAGE, Native-PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecular mass of ∼5.5 and 7.0 kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis.

    PubMed

    Jakob, Virginie; Brunner, Livia; Barnier-Quer, Christophe; Blust, Molly; Collin, Nicolas; Carter, Lauren; Carter, Darrick; Rausch, Kelly M; Fox, Christopher B

    2017-04-01

    Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. These methods may be useful for improving characterization approaches of antigen-adjuvant mixtures by SDS-PAGE.

  8. Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis.

    PubMed

    Temmerman, R; Scheirlinck, I; Huys, G; Swings, J

    2003-01-01

    In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.

  9. Microfluidic polyacrylamide gel electrophoresis with in situ immunoblotting for native protein analysis.

    PubMed

    He, Mei; Herr, Amy E

    2009-10-01

    We introduce an automated immunoblotting method that reports protein electrophoretic mobility and identity in a single streamlined microfluidic assay. Native polyacrylamide gel electrophoresis (PAGE) was integrated with subsequent in situ immunoblotting. Integration of three PA gel elements into a glass microfluidic chip achieved multiple functions, including (1) rapid protein separation via on-chip PAGE, (2) directed electrophoretic transfer of resolved protein peaks to an in-line blotting membrane, and (3) high-efficiency identification of the transferred proteins using antibody-functionalized blotting membranes. In-chip blotting membranes were photopatterned with biotinylated antibody using streptavidin polyacrylamide (PA) thus yielding postseparation sample analysis. No pressure driven flow or fluid valving was required, as the assay was operated by electrokinetically programmed control. A model sample of fluorescently labeled BSA (negative control), alpha-actinin, and prostate specific antigen (PSA) was selected to develop and characterize the assay. A 5 min assay time was required without operator intervention. Optimization of the blotting membrane (geometry, operation, and composition) yielded a detection limit of approximately 0.05 pg (alpha-actinin peak). An important additional blotting fabrication strategy was developed and characterized to allow vanishingly small antibody consumption (approximately 1 microg), as well as end-user customization of the blotting membrane after device fabrication and storage. This first report of rapid on-chip protein PAGE integrated with in situ immunoblotting forms the basis for a sensitive, automated approach applicable to numerous forms of immunoblotting.

  10. Nested PCR-denaturing gradient gel electrophoresis analysis of human skin microbial diversity with age.

    PubMed

    Li, Wei; Han, Lei; Yu, Pengbo; Ma, Chaofeng; Wu, Xiaokang; Xu, Jiru

    2014-01-01

    To determine whether the composition and structure of skin microbiota differ with age, cutaneous bacteria were isolated from the axillary fossa of 37 healthy human adults in two age groups (old people and young adults). Bacterial genomic DNA was extracted and characterized by nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region of the 16S rRNA gene. The excised gel bands were sequenced to identify bacterial categories. The total bacteria, Staphylococcus spp., Staphylococcus epidermidis and Corynebacterium spp. were further enumerated by quantitative PCR. There were no significant differences in the species diversity profiles between age groups. The similarity index was lower across age groups than that it was intra-group. This indicates that the composition of skin flora is more similar to others of the same age than across age groups. While Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria in both groups, sequencing and quantitative PCR revealed that skin bacterial composition differed by age. The copy number of total bacteria and Corynebacterium spp. were significantly lower in younger subjects, whereas there were no statistical differences in the quantity of Staphylococcus spp. and Staphylococcus epidermidis. These results suggest that the skin flora undergo both quantitative and qualitative changes related to aging.

  11. Urine proteins identified by two-dimensional differential gel electrophoresis facilitate the differential diagnoses of scrapie.

    PubMed

    Lamoureux, Lise; Simon, Sharon L R; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.

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

    PubMed Central

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    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. PMID:27092510

  13. Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis

    PubMed Central

    Jakob, Virginie; Brunner, Livia; Barnier-Quer, Christophe; Blust, Molly; Collin, Nicolas; Carter, Lauren; Carter, Darrick; Rausch, Kelly M.; Fox, Christopher B.

    2017-01-01

    Objectives: Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. Methods: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. Results: The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. Conclusions: These methods may be useful for improving characterization approaches of antigen–adjuvant mixtures by SDS-PAGE. PMID:28515938

  14. Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining.

    PubMed

    Zhao, Dong S; Roy, Binayak; McCormick, Matthew T; Kuhr, Werner G; Brazill, Sara A

    2003-05-01

    In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1).

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

    PubMed

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-04-16

    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.

  16. A novel multi-scale Hessian based spot enhancement filter for two dimensional gel electrophoresis images.

    PubMed

    Shamekhi, Sina; Miran Baygi, Mohammad Hossein; Azarian, Bahareh; Gooya, Ali

    2015-11-01

    Two dimensional gel electrophoresis (2DGE) is a useful method for studying proteins in a wide variety of applications including identifying post-translation modification (PTM), biomarker discovery, and protein purification. Computerized segmentation and detection of the proteins are the two main processes that are carried out on the scanned image of the gel. Due to the complexities of 2DGE images and the presence of artifacts, the segmentation and detection of protein spots in these images are non-trivial, and involve supervised and time consuming processes. This paper introduces a new spot filter for enhancing, and separating the closely overlapping spots of protein in 2DGE images based on the multi-scale eigenvalue analysis of the image Hessian. Using a Gaussian spot model, we have derived closed form equations to compute the eigen components of the image Hessian of two overlapping spots in a multi-scale fashion. Based on this analysis, we have proposed a novel filter that suppresses the overlapping area and results in a better spot separation. The performance of the proposed filter has been evaluated on the synthetic and real 2DGE images. The comparison with three conventional techniques and a commercial software package reveals the superiority and effectiveness of the proposed filter.

  17. Theory of DNA electrophoresis in physical gels and entangled polymer solutions

    NASA Astrophysics Data System (ADS)

    Duke, Thomas; Viovy, Jean Louis

    1994-03-01

    A scaling theory is presented for the electrophoretic mobility of DNA in sieving media that form dynamically evolving meshworks, such as physical gels and solutions of entangled polymers. In such media, the topological constraints on the DNA's motion are perpetually changing as cross links break and rejoin or as the polymers diffuse. It is shown that if the rate of constraint release falls within a certain range (which depends on the field strength), fractionation can be extended to higher molecular weights than would be feasible using a permanent gel of equivalent pore size. This improvement is a consequence of the disruptive effect that constraint release has on the mechanism of molecular orientation. Numerical simulations support the predictions of the theory. The possibility of realizing such a system in practice, with the aim of improving on current electrophoresis methods, is commented upon. It is suggested that semidilute polymer solutions may be a versatile medium for the rapid separation of long single-stranded DNA molecules, and the particular quality of solution required is identified.

  18. Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis.

    PubMed

    Zuobi-Hasona, Kheir; Crowley, Paula J; Hasona, Adnan; Bleiweis, Arnold S; Brady, L Jeannine

    2005-03-01

    Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.

  19. Serum alkaline phosphatase isoenzymes in laboratory beagle dogs detected by polyacrylamide-gel disk electrophoresis.

    PubMed

    Hatayama, Kazuhisa; Nishihara, Yoshito; Kimura, Sayaka; Goto, Ken; Nakamura, Daichi; Wakita, Atsushi; Urasoko, Yoshinaka

    2011-10-01

    Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. Itoh et al. (2002) reported that a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes was useful for veterinary clinicopathological diagnosis in mongrel dogs. In the present study, based on the report of Itoh et al. (2002), we tried to expand the application range of this kit to laboratory beagle dogs which are commonly used in toxicity studies. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine and serum samples were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The main serum ALP isoenzymes in 5-month-old intact beagle dogs were bone-derived (bone and atypical ALP: corresponding to human variant bone ALP) and they tended to decrease with age. However, liver-derived ALP isoenzyme greatly increased in the serum of cholestasis model dogs. The cholestasis model dogs also had a large molecular ALP detected in the resolving gel. This ALP could be originated from intestinal ALP or corticosteroid-induced ALP (CALP), because the activity remained even after levamisole inhibition. CALP was observed in intact laboratory beagle dogs with individual differences. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in laboratory beagle dogs and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

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

    PubMed

    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.

  1. A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens.

    PubMed

    Levy, D; Davies, J; O'Hehir, R; Suphioglu, C

    2001-06-01

    Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 microg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

  2. Stabilization of thin-layer agarose gels after isoelectric focusing with polyacrylamide enables reverse imidazole-zinc staining and facilitates two-dimensional gel electrophoresis.

    PubMed

    Hellman, Jukka

    2008-09-01

    Large-pore-size agarose gels provide excellent resolving capacity for high molecular weight biomolecules. Thin-layer agarose isoelectric focusing (IEF) gels on polyester support films are especially useful for the separation of large proteins based on their pI in native conformation, but the method has suffered from the lack of detection methods compatible with agarose gels in hydrated form. Recently, an acrylamide copolymerization method was reported to enable mass-spectrometry-compatible silver staining and in-gel digestion of proteins. In this study, the method was further applied by demonstrating successful reverse imidazole-zinc staining of thin-layer agarose IEF gels copolymerized with acrylamide. The sensitivity of the reverse staining method on the composite gel at its best equaled the sensitivity of the traditional dried agarose silver staining method. Owing to the increased durability and reversible detection, the reverse-stained first-dimension gel could be conveniently prepared for the second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis by reduction and alkylation. In addition, the micropreparative generation of tryptic peptides of Coomassie brilliant blue R-250 stained proteins in the composite gel is demonstrated.

  3. The basics of 2D DIGE.

    PubMed

    Beckett, Phil

    2012-01-01

    The technique of two-dimensional (2D) gel electrophoresis is a powerful tool for separating complex mixtures of proteins, but since its inception in the mid 1970s, it acquired the stigma of being a very difficult application to master and was generally used to its best effect by experts. The introduction of commercially available immobilized pH gradients in the early 1990s provided enhanced reproducibility and easier protocols, leading to a pronounced increase in popularity of the technique. However gel-to-gel variation was still difficult to control without the use of technical replicates. In the mid 1990s (at the same time as the birth of "proteomics"), the concept of multiplexing fluorescently labeled proteins for 2D gel separation was realized by Jon Minden's group and has led to the ability to design experiments to virtually eliminate gel-to-gel variation, resulting in biological replicates being used for statistical analysis with the ability to detect very small changes in relative protein abundance. This technology is referred to as 2D difference gel electrophoresis (2D DIGE).

  4. GEL-STATE NMR OF BALL-MILLED WHOLE CELL WALLS IN DMSO-d6 USING 2D SOLUTION-STATE NMR SPECTROSCOPY

    USDA-ARS?s Scientific Manuscript database

    Plant cell walls were used for obtaining 2D solution-state NMR spectra without actual solubilization or structural modification. Ball-milled whole cell walls were swelled directly in the NMR tube with DMSO-d6 where they formed a gel. There are relatively few gel-state NMR studies. Most have involved...

  5. Seeing through the trick of cancer cells via 2D gels.

    PubMed

    Mao, Lei

    2013-12-01

    The advancement of modern therapy concepts has dramatically extended the postsurvival rates of patients with malignant gastric cancer. However, a remaining setback is the drug resistance of recurrent cancer, which casts a dark shadow over disease prognosis. The original work of Klein et al. [Proteomics Clin. Appl. 2013, 7, 813-824] has outlined a rational experimental approach to decipher the mechanistic pathway of cancer drug resistance by proteomic approach. They used gel-based comparative proteomics to analyze the nuclear proteome of a human gastric cancer cell line (AGS) with and without inactivation of hypoxia-inducible factor 1 (HIF-1), a transcription factor and master regulator of hypoxia adaptation. Using the classical 2DE-MS approach, these researchers observed 163 HIF-1 responsive proteins, among which over half of them could be confidently identified by MS. From this large dataset, the authors proposed an enhanced nuclear translocation of some proteasomal proteins upon inactivation of HIF-1. Overall, this work appropriately used proteomics as a hypothesis-free, top-down approach to dissect imperative clinical problems.

  6. Disease proteomics of high-molecular-mass proteins by two-dimensional gel electrophoresis with agarose gels in the first dimension (Agarose 2-DE).

    PubMed

    Oh-Ishi, Masamichi; Maeda, Tadakazu

    2007-04-15

    Agarose gel is the preferred electrophoretic medium currently used for separating high molecular mass (HMM) proteins (MW>100 kDa). Agarose gels are widely used for both SDS-agarose gel electrophoresis and agarose isoelectric focusing (IEF). A two-dimensional gel electrophoresis method employing agarose gels in the first dimension (agarose 2-DE) that is sufficiently good at separating up to 1.5mg of HMM proteins with molecular masses as large as 500 kDa has been used to separate proteins from various diseased tissues and cells. Although resolution of the agarose 2-DE pattern always depends on the tissue being analyzed, sample preparation procedures including (i) protein extraction with an SDS sample buffer; (ii) ultracentrifugation of a tissue homogenate; and (iii) 1% SDS in both stacking and separation gels of the second-dimension SDS-PAGE gel, are generally effective for HMM protein detection. In a comprehensive prostate cancer proteome study using agarose 2-DE, the HMM region of the gel was rich in proteins of particular gene/protein expression groups (39.1% of the HMM proteins but only 28.4% of the LMM ones were classified as transcription/translation-related proteins). Examples include transcription factors, DNA or RNA binding proteins, and ribosomal proteins. To understand oxidative stress-induced cellular damage at the protein level, a novel proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by agarose 2-DE, was useful for detecting HMM protein carbonyls in tissues of both a diabetes model Ostuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. In this paper, we review the use of agarose gels for separation of HMM proteins and disease proteomics of HMM proteins in general, with particular attention paid to our proteome analyzes based on the use of agarose 2-DE for protein separation followed by the use of mass spectrometry for protein identification.

  7. Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene.

    PubMed

    Elek, Zsuzsanna; Dénes, Réka; Prokop, Susanne; Somogyi, Anikó; Yowanto, Handy; Luo, Jane; Souquet, Manfred; Guttman, András; Rónai, Zsolt

    2016-09-01

    The WFS1 gene is one of the thoroughly investigated targets in diabetes research, variants of the gene were suggested to be the genetic components of the common forms (type 1 and type 2) of diabetes. Our project focused on the analysis of polymorphisms (rs4689388, rs148797429, rs4273545) localized in the WFS1 promoter region. Although submarine gel electrophoresis based approaches were also employed in the genetic tests, it was demonstrated that multicapillary electrophoresis offers a state of the art approach for reliable high-throughput SNP and VNTR analysis. Association studies were carried out in a case-control setup. Luciferase reporter assay was employed to test the effect of the investigated loci on the activity of gene expression in vitro. Significant association could be demonstrated between all three polymorphisms and type 2 diabetes in both allele- and genotype-wise settings even using Bonferroni correction. It is notable; however, that the three loci were in strong linkage disequilibrium, thus the observed associations cannot be considered as separate effects. Molecular analyses showed that the rs4273545 GT SNP played a role in the regulation of transcription in vitro. However, this effect took place only in the presence of the region including the rs148797429 site, although this latter locus did not have its own impact on the regulation of gene expression. The paper provides genotyping protocols readily applicable in any multiplex SNP and VNTR analyses, moreover confirms and extends previous results about the role of WFS1 polymorphisms in the genetic risk of diabetes mellitus.

  8. A new method combining sequential immunoaffinity depletion and differential in gel electrophoresis to identify autoantibodies as cancer biomarkers.

    PubMed

    Grandjean, Marie; Dieu, Marc; Raes, Martine; Feron, Olivier

    2013-10-31

    Easily measurable biomarkers are urgently required to detect early stages of cancer progression. Autoantibodies (aAbs), as a component of the humoral immune response against tumor cells, have such potential of diagnostic markers since they are circulating and stable proteins, produced rapidly and easily amenable to in vitro dosage. The identification of aAbs is based on the characterization of tumor-associated antigens (TAA) against which they are directed. Here, we propose a new method for an unbiased identification of TAA and thereby of aAbs as cancer biomarkers. This method that we called sequential immunoaffinity depletion-differential in gel electrophoresis (SID-DIGE) is based on the immunodepletion of tumor cell lysates with IgG from control and tumor-bearing mice and direct matching of the flow throughs of these immunoaffinity separations on the same 2D format. This strategy reduces the complexity of the samples to be analyzed and maximizes the interest of assessing hundreds of proteins simultaneously. SID-DIGE has also the potential, contrary to existing serological proteome analysis (SERPA) techniques, to detect immunogenic proteins with conformational epitopes, including those resulting from post-translational modifications. Using a model of human colorectal tumors in mice for the proof of principle, we showed that SID-DIGE outperforms the conventional SERPA technique, with the identification of 7 common TAA (validating our approach) and 18 additional aAbs proving the potential of this new method. In particular, the identification of aAbs directed against key enzymes supporting glycolysis gives credential to the role of hypoxia as a major determinant of the tumor proteome and thus as a source of immunogenicity. Overall, the developed methodology allowed efficient screening of sera for the identification of aAbs as potential biomarkers.

  9. Two-Dimensional Differential Gel Electrophoresis to Identify Protein Biomarkers in Amniotic Fluid of Edwards Syndrome (Trisomy 18) Pregnancies

    PubMed Central

    Hsu, Te-Yao; Lin, Hao; Hung, Hsuan-Ning; Yang, Kuender D.; Ou, Chia-Yu; Tsai, Ching-Chang; Cheng, Hsin-Hsin; Chung, Su-Hai; Cheng, Bi-Hua; Wong, Yi-Hsun; Chou, An Kuo; Hsiao, Chang-Chun

    2016-01-01

    Background Edwards syndrome (ES) is a severe chromosomal abnormality with a prevalence of about 0.8 in 10,000 infants born alive. The aims of this study were to identify candidate proteins associated with ES pregnancies from amniotic fluid supernatant (AFS) using proteomics, and to explore the role of biological networks in the pathophysiology of ES. Methods AFS from six second trimester pregnancies with ES fetuses and six normal cases were included in this study. Fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were used for comparative proteomic analysis. The identified proteins were further validated by Western blotting and the role of biological networks was analyzed. Results Twelve protein spots were differentially expressed by more than 1.5-fold in the AFS of the ES pregnancies. MALDI-TOF/MS identified one up-regulated protein: apolipoprotein A1 (ApoA1), and four under-regulated proteins: vitamin D binding protein (VDBP), alpha-1-antitrypsin (A1AT), insulin-like growth factor-binding protein 1 (IGFBP-1), and transthyretin (TTR). Western blot and densitometric analysis of ApoA1, A1AT, IGFBP-1, and TTR confirmed the alteration of these proteins in the amniotic fluid samples. Biological network analysis revealed that the proteins of the ES AFS were involved mainly in lipid and hormone metabolism, immune response, and cardiovascular disease. Conclusions These five proteins may be involved in the pathogenesis of ES. Further studies are needed to explore. PMID:26752631

  10. Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

    PubMed

    Mears, Rainy; Craven, Rachel A; Hanrahan, Sarah; Totty, Nick; Upton, Carol; Young, Sarah L; Patel, Poulam; Selby, Peter J; Banks, Rosamonde E

    2004-12-01

    Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti

  11. Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses[S

    PubMed Central

    Toledo-Ibelles, Paola; García-Sánchez, Cynthia; Ávila-Vazzini, Nydia; Carreón-Torres, Elizabeth; Posadas-Romero, Carlos; Vargas-Alarcón, Gilberto; Pérez-Méndez, Oscar

    2010-01-01

    The aim of this study was to develop an enzymatic cholesterol staining method to determine HDL subclasses in a polyacrylamide gradient gel electrophoresis, which further allows staining by protein in the same electrophoresis lane. HDLs from 120 healthy individuals were separated through nondenaturing PAGE. HDLs were stained for cholesterol using an enzymatic semisolid mixture. Once the gels were unstained, they were stained again for proteins with Coomassie blue. The proportions of HDL subclasses were determined by densitometry. HDL subclasses were transformed to concentrations using as reference HDL-cholesterol plasma levels. This method is comparable in linearity and reproducibility to Coomassie blue staining, although it provides quantitative data. As expected, HDL size distribution shifted toward larger particles when determined by cholesterol as compared with protein. With this method, we observed different proportions of HDL subclasses between men and women as compared with Coomassie blue staining. We described a method to determine HDL size distribution by enzymatic cholesterol staining on polyacrylamide gels. The method allows the quantification of the cholesterol plasma concentration of each HDL subclass with the possibility to further stain the protein in the same sample. The combination of HDL staining by cholesterol and protein on electrophoresis gels provides information that may have clinical relevance. PMID:20097938

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

    PubMed

    Choi, Megan; Nordmeyer, Robert A; Cornell, Earl; Dong, Ming; Biggin, Mark D; Jin, Jian

    2010-01-01

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.

  13. The use of biphasic linear ramped pulsed field gel electrophoresis to quantify DNA damage based on fragment size distribution

    SciTech Connect

    Lawrence, T.S.; Normolle, D.P.; Davis, M.A.; Maybaum, J.

    1993-10-20

    The development of biphasic linear pulse ramping gel electrophoresis has permitted resolution of DNA fragments from 200 Kbp to 6 Mbp in a single gel. We used this technique to measure radiation-induced DNA damage based on fragment size. Human colon cancer cells (HT29 and LS174T) and Chinese hamster ovary cells were embedded in agarose, deproteinized, irradiated with 5-80 Gy, and assessed for DNA double strand breakage using pulsed field gel electrophoresis. The frequency of DNA double strand breakage determined using a previously published method was compared to the breakage frequency calculated using the fragment size distribution. Both methods produced similar estimates for breakage frequency of approximately 5 {times} 10{sup {minus}9} breaks Gy{sup {minus}1} bp{sup {minus}1}. These findings suggest that biphasic linear pulse ramping gel electrophoresis can yield a quantitative estimate of DNA fragment distribution resulting from irradiation. The ability to quantify the distribution of DNA fragment sizes produced by irradiation should yield information concerning the mechanisms of both DNA double strand break induction and repair. 16 refs., 5 figs.

  14. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    SciTech Connect

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

  15. Detection of enzymes active on various beta-1,3-glucans after denaturing polyacrylamide gel electrophoresis.

    PubMed

    Trudel, J; Grenier, J; Asselin, A

    1998-07-01

    Enzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing beta-1,3-glucans embedded as substrate. Lentinan, curdlan, paramylon, baker's yeast alkali-insoluble glucan, baker's yeast alkali-soluble glucan and carboxymethyl (CM)-pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying beta-1,3-glucanase activities. Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5-triphenyltetrazolium chloride (TTC). For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures. No Lyticase enzyme was detected in either detection procedures for all tested substrates. For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM-pachyman as substrates. In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures. This was the same for the barley leaf extract (32, 36 and 39 kDa). The Driselase extract showed one 42 kDa band. Many enzymes active on beta-1,3-glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS-PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers. However, some enzymes are only detected when proteins are denatured without reduction. Finally, the use of various polymeric beta-1,3-glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.

  16. Microbial Community Structure of Korean Cabbage Kimchi and Ingredients with Denaturing Gradient Gel Electrophoresis.

    PubMed

    Hong, Sung Wook; Choi, Yun-Jeong; Lee, Hae-Won; Yang, Ji-Hee; Lee, Mi-Ai

    2016-06-28

    Kimchi is a traditional Korean fermented vegetable food, the production of which involves brining of Korean cabbage, blending with various other ingredients (red pepper powder, garlic, ginger, salt-pickled seafood, etc.), and fermentation. Recently, kimchi has also become popular in the Western world because of its unique taste and beneficial properties such as antioxidant and antimutagenic activities, which are derived from the various raw materials and secondary metabolites of the fermentative microorganisms used during production. Despite these useful activities, analysis of the microbial community present in kimchi has received relatively little attention. The objective of this study was to evaluate the bacterial community structure from the raw materials, additives, and final kimchi product using the culture-independent method. Specifically, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the 16S rRNA partial sequences of the microflora. One primer set for bacteria, 341F(GC)-518R, reliably produced amplicons from kimchi and its raw materials, and these bands were clearly separated on a 35-65% denaturing gradient gel. Overall, 117 16S rRNA fragments were identified by PCR-DGGE analysis. Pediococcus pentosaceus, Leuconostoc citreum, Leuconostoc gelidum, and Leuconostoc mesenteroides were the dominant bacteria in kimchi. The other strains identified were Tetragenococcus, Pseudomonas, Weissella, and uncultured bacterium. Comprehensive analysis of these microorganisms could provide a more detailed understanding of the biologically active components of kimchi and help improve its quality. PCR-DGGE analysis can be successfully applied to a fermented food to detect unculturable or other species.

  17. Genetic fingerprinting of Brevibacterium linens by pulsed-field gel electrophoresis and ribotyping.

    PubMed

    Lima, P T; Correia, A M

    2000-07-01

    Members of Brevibacterium linens display physiological features that are relevant for cheese production. The genomes of five B. linens strains deposited on culture collections were compared by examining large restriction fragments on pulsed-field gel electrophoresis and detection of polymorphism at the level of 16S rRNA genes. Pulsed-field analysis with the endonucleases DraI and AsnI showed a characteristic restriction profile for each strain and allowed the calculation of genome sizes ranging between 3.2 and 3.9 Mbp. No linear genomic elements were detected. Polymorphisms at the level of 16S rRNA genes were revealed by hybridization with an oligonucleotide probe complementary to a universal domain of the 16S genes. An EcoRI fragment of 1.4 kb was identified as common to all strains under study. According to the number of positive bands detected by the probe, at least four rRNA operons must be present on the genome of the B. linens strains here studied.

  18. Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

    PubMed Central

    Satokari, Reetta M.; Vaughan, Elaine E.; Akkermans, Antoon D. L.; Saarela, Maria; de Vos, Willem M.

    2001-01-01

    We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074T exhibited microheterogeneity differing in eight positions over almost the total length of the gene. PMID:11157210

  19. Electrochemical detection of interaction between capsaicin and nucleic acids in comparison to agarose gel electrophoresis.

    PubMed

    Yilmaz, Nilay; Eksin, Ece; Karacicek, Bilge; Eraç, Yasemin; Erdem, Arzum

    2017-10-15

    In this study, the biomolecular interaction occurred between nucleic acids and Capsaicin (CPS), the active compound in chilli peppers, which has been reported to have anti-carcinogenic properties, was investigated for the first time herein using disposable electrochemical biosensor. It is aimed to perform the surface-confined interaction between CPS and different types of nucleic acids and under this aim, the experimental conditions were optimized; such as, the concentration of CPS and DNA, DNA immobilization time and interaction time etc. The detection limit of DNA was estimated based on guanine oxidation signal in the linear concentration range of DNA from 1 to 5 μg/mL, and it was found to be 0.62 μg/mL. The effect of time-dependent manner from 1 min to 30 min on the interaction of CPS with nucleic acids was explored upon to the changes at guanine signal coming from double stranded DNA and cDNA as well as PCR samples. The interaction of CPS with double stranded DNA was also determined by agarose gel electrophoresis. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Difference gel electrophoresis (DiGE) identifies differentially expressed proteins in endoscopically-collected pancreatic fluid

    PubMed Central

    Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

    2012-01-01

    Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

  1. Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis

    PubMed Central

    Janssen, Brian D.; Diner, Elie J.; Hayes, Christopher S.

    2013-01-01

    During protein synthesis, the ribosome translates the genetic information encoded within messenger RNAs into defined amino acid sequences. Transfer RNAs (tRNAs) are crucial adaptor molecules in this process, delivering amino acid residues to the ribosome and holding the nascent peptide chain as it is assembled. Here, we present methods for the analysis of aminoacyl- and peptidyl-tRNA species isolated from Escherichia coli. These approaches utilize denaturing gel electrophoresis at acidic pH to preserve the labile ester bonds that link amino acids to tRNA. Specific aminoacyl- and peptidyl-tRNAs are detected by Northern blot hybridization using probes for tRNA isoacceptors. Small peptidyl-tRNAs can be differentiated from aminoacyl-tRNA through selective deacylation of the latter with copper sulfate. Additionally, peptidyl-tRNAs can be detected through metabolic labeling of the nascent peptide. This latter approach is amenable to pulse-chase analysis to examine peptidyl-tRNA turnover in vivo. We have applied these methods to study programmed translational arrests and the kinetics of paused ribosome turnover. PMID:22736012

  2. Prediction System for Rapid Identification of Salmonella Serotypes Based on Pulsed-Field Gel Electrophoresis Fingerprints

    PubMed Central

    Lin, Wei-Jiun; Hise, Kelley B.; Chen, Hung-Chia; Keys, Christine; Chen, James J.

    2012-01-01

    A classification model is presented for rapid identification of Salmonella serotypes based on pulsed-field gel electrophoresis (PFGE) fingerprints. The classification model was developed using random forest and support vector machine algorithms and was then applied to a database of 45,923 PFGE patterns, randomly selected from all submissions to CDC PulseNet from 2005 to 2010. The patterns selected included the top 20 most frequent serotypes and 12 less frequent serotypes from various sources. The prediction accuracies for the 32 serotypes ranged from 68.8% to 99.9%, with an overall accuracy of 96.0% for the random forest classification, and ranged from 67.8% to 100.0%, with an overall accuracy of 96.1% for the support vector machine classification. The prediction system improves reliability and accuracy and provides a new tool for early and fast screening and source tracking of outbreak isolates. It is especially useful to get serotype information before the conventional methods are done. Additionally, this system also works well for isolates that are serotyped as “unknown” by conventional methods, and it is useful for a laboratory where standard serotyping is not available. PMID:22378901

  3. Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis.

    PubMed

    Endo, Akihito; Okada, Sanae

    2005-03-01

    The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.

  4. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

    PubMed

    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.

  5. The limitations of pulsed-field gel electrophoresis for analysis of Yersinia enterocolitica isolates.

    PubMed

    Gilpin, B J; Robson, B; Lin, S; Hudson, J A; Weaver, L; Dufour, M; Strydom, H

    2014-09-01

    This study describes the analysis of 432 isolates of Yersinia enterocolitica by pulsed-field gel electrophoresis (PFGE). PFGE had a high level of discrimination with biotype 1A isolates (Simpson's Diversity Index 0.997), but with the clinically important biotypes 2, 3 and 4, the discriminatory ability of PFGE was so low as to severely limit its usefulness (DI <0.6). For biotypes 2, 3 and 4, 79% or more of isolates of each biotype were of just three different PFGE profiles. Because of this, four known outbreaks of yersiniosis would not have been identified by PFGE analysis. However, a previously unrecognized potential outbreak of yersiniosis caused by biotype 4 isolates was identified on the basis of a rare PFGE genotype with spatial and temporal clustering. We conclude that PFGE has a very limited application to the genotyping of Y. enterocolitica biotypes 2, 3 and 4, and inferences based on finding indistinguishable PFGE profiles among cases or between cases and sources need to be substantiated using alternative typing tools, or strong epidemiological evidence.

  6. Polyacrylamide gel disc electrophoresis of alkaline phosphatase isoenzymes in bone and liver disease.

    PubMed Central

    Warnes, T W; Hine, P; Kay, G

    1976-01-01

    Acrylamide gel disc electrophoresis provides a reliable and reasonably rapid method of differentiating the raised serum alkaline phosphatase (AP) of bone origin from that of liver origin. The technique has been placed for the first time on a semiquantitative basis. Measurement of both band width and band position effectively distinguishes the bone from the liver isoenzyme, but band width provides superior discrimination. An origin band was seen in none of the normal subjects and in only 7% of patients with bone disease but was present in 78% of patients with liver disease, a highly significant increase. Fifty percent of normal individuals had a small-intestinal band in serum taken two hours after a meal, as did 35% of patients with liver disease, but the incidence of intestinal bands in bone disease was only 11%, significantly less than in the other two groups. The genetic control of small-intestinal AP in serum has been confirmed, but it has been demonstrated that the decrease of intestinal AP in bone disorders is not genetically determined. Images PMID:977779

  7. Pulsed-field gel electrophoresis and ribotype profiles of clinical and environmental Vibrio vulnificus isolates.

    PubMed Central

    Tamplin, M L; Jackson, J K; Buchrieser, C; Murphree, R L; Portier, K M; Gangar, V; Miller, L G; Kaspar, C W

    1996-01-01

    Vibrio vulnificus belongs to the autochthonous bacterial flora of warm estuarine waters. It can cause life-threatening extraintestinal disease in persons who have underlying illness and who consume raw shellfish or contact wounds with estuarine water. Currently, very little is known about genetic diversity within this species. In this report, we describe high-level variation in restriction fragment length polymorphism profiles among 53 clinical and 78 environmental isolates, as determined by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. When combined ribotype profiles of clinical and environmental isolates were analyzed, four predominant clusters were observed. Interestingly, a low number (16%) of clinical isolates were found in cluster C, compared with clusters A, B, and D (range, 50 to 83%). In addition, 83% of all Hawaiian isolates were located in a single cluster, indicating a possible relationship between geography and genotype. We also report that spontaneous translucent colonial morphotypes were distinct by both restriction fragment length polymorphism and biochemical profiles, compared with opaque parent strains. PMID:8837412

  8. Epidemiological Validation of Pulsed-Field Gel Electrophoresis Patterns for Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Blanc, D. S.; Struelens, M. J.; Deplano, A.; De Ryck, R.; Hauser, P. M.; Petignat, C.; Francioli, P.

    2001-01-01

    To determine the stability of pulsed-field gel electrophoresis (PFGE) patterns of methicillin-resistant Staphylococcus aureus in the nosocomial setting, we analyzed isolates from long-term carriers (>1 month) and from patients involved in well-defined nosocomial epidemics. The number of fragment differences between the first isolate and subsequent isolates in long-term carriers showed a bimodal distribution, with one group having 0 to 6 fragment differences and the other group having 14 to 24 fragment differences. The PFGE patterns of isolates involved in epidemics also presented a similar bimodal distribution of the number of fragment differences. Typing these isolates with another molecular method (inter-IS256 PCR) showed that isolates of the first group (i.e., with 1 to 6 fragment differences) were clonally related, whereas the second group (with 14 to 24 fragment differences) could be considered genetically different. Among long-term carriers with clonally related isolates, 74 of 84 (88%) of consecutive isolates showed indistinguishable patterns, whereas 10 of 84 (12%) showed related patterns differing by one to six fragments. Moreover, the frequency of apparition of related patterns is higher when the time between the first and the subsequent isolate is longer. During seven nosocomial epidemics lasting from 1 to 15 months, only 2 of 120 isolates (1.7%) showed a pattern which was different, although related, from the predominant one involved in each of these outbreaks. PMID:11574553

  9. Assessment of microbial populations dynamics in a blue cheese by culturing and denaturing gradient gel electrophoresis.

    PubMed

    Alegría, Angel; González, Renata; Díaz, Mario; Mayo, Baltasar

    2011-03-01

    The composition and development of microbial population during the manufacture and ripening of two batches of a blue-veined cheese was examined by culturing and polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE). Nine selective and/or differential media were used to track the cultivable populations of total and indicator microbial groups. For PCR-DGGE, the V3 hyper variable region of the bacterial 16S rRNA gene and the eukaryotic D1 domain of 28S rDNA were amplified with universal primers, specific for prokaryotes and eukaryotes, respectively. Similarities and differences between the results obtained by the culturing and the molecular method were recorded for some populations. Culturing analysis allows minority microbial groups (coliforms, staphylococci) to be monitored, although in this study PCR-DGGE identified a population of Streptococcus thermophilus that went undetected by culturing. These results show that the characterization of the microbial populations interacting and evolving during the cheese-making process is improved by combining culturing and molecular methods.

  10. Brownian dynamics studies on DNA gel electrophoresis. II. ``Defect'' dynamics in the elongation-contraction motion

    NASA Astrophysics Data System (ADS)

    Azuma, Ryuzo

    2002-10-01

    By means of the Brownian dynamics (BD) method of simulations we have developed, we study the dynamics of individual DNA molecules which are undergoing constant field gel electrophoresis (CFGE), focusing on the relevance of the "defect" concept due to de Gennes in CFGE. The corresponding objects, which we call slack beads (s-beads), are explicitly introduced in our BD model. In equilibrium under a vanishing field, the distance between s-beads and their hopping range is found to be randomly distributed following a Poisson distribution. In strong fields, where a chain undergoes elongation-contraction motion, s-beads are observed to be alternately annihilated in elongation and created in the contraction of the chain. On the other hand, the distribution of hopping ranges of s-beads does not differ much from that in equilibrium. The results indicate that in the elongation-contraction motion of the chain, a large number of random movements of s-beads are involved. We have also confirmed that these features of s-beads agree qualitatively with those of s-monomers in the extended bond fluctuation model (EBFM) which we recently proposed. This agreement strongly supports the stochastic semilocal movement of s-monomers which we a priori introduced into the EBFM.

  11. Typing of Methicillin Resistant Staphylococcus Aureus Using DNA Fingerprints by Pulsed-field Gel Electrophoresis

    PubMed Central

    Rebic, Velma; Budimir, Ana; Aljicevic, Mufida; Bektas, Sabaheta; Vranic, Sabina Mahmutovic; Rebic, Damir

    2016-01-01

    Background: Methicillin resistant Staphylococcus aureus (MRSA) is responsible for a wide spectrum of nosocomial and community associated infections worldwide. The aim of this study was to analyze MRSA strains from the general population in Canton Sarajevo, B&H. Methods: Our investigation including either phenotypic and genotypic markers such as antimicrobial resistance, pulsed-field gel electrophoresis (PFGE), SCC typing, and Panton-Valentine leukocidin (PVL) detection. Results: Antimicrobial susceptibility: all MRSA isolates were resistant to the β-lactam antibiotics tested, and all isolates were susceptible trimethoprim sulphamethoxazole, rifampicin, fusidic acid, linezolid and vancomycin. Sixty-eight per cent of the MRSA isolates were resistant to erythromycin, 5% to clindamycin, 5% to gentamicin and 4% to ciprofloxacin. After the PFGE analysis, the isolates were grouped into five similarity groups: A-E. The largest number of isolates belonged to one of two groups: C: 60 (60%) and D: 27 (27%). In both groups C and D, SCCmec type IV was predominant (60% and 88, 8%, respectively). A total of 24% of the isolates had positive expression of PVL genes, while 76% showed a statistically significantly greater negative expression of PVL genes. Conclusion: SCCmec type IV, together with the susceptibility profile and PFGE grouping, is considered to be typical of CA-MRSA PMID:27708486

  12. Application of pulsed-field gel electrophoresis to identify potential outbreaks of campylobacteriosis in New Zealand.

    PubMed

    Gilpin, Brent; Cornelius, Angela; Robson, Beth; Boxall, Naomi; Ferguson, Alan; Nicol, Carolyn; Henderson, Tom

    2006-02-01

    Since 2002, New Zealand's incidence of campylobacteriosis has exceeded 300 cases per 100,000 people per annum. To evaluate genetic variation in human isolates, 183 Campylobacter isolates were collected from a single clinical laboratory in Christchurch: 77 during an 8-week period in spring, and the rest 3 months later over a second 8-week period in autumn. Isolates were identified to the species level and subtyped using Penner serotyping (Campylobacter jejuni only) and pulsed-field gel electrophoresis (PFGE) using both SmaI and KpnI. Approximately two-thirds of the isolates could be grouped into clusters of between 2 and 26 isolates with indistinguishable SmaI and KpnI patterns. Less than 10% of the isolates were of the same type between the two sampling periods. The epidemiological relevance of the PFGE clusters was supported by temporal clustering, some spatial clustering, and some statistically significant demographic similarities among cases in a cluster. Conversely, patient cases yielding isolates which did not cluster with isolates from other cases were more likely to report recent overseas travel and less likely to live within larger urban centers. To identify whether these clusters actually represent common-source outbreaks, however, would require the detailed, rapid, and reiterative epidemiological investigation of cases within a PFGE cluster. The combined and timely application of subtyping and epidemiological investigation would appear to be a promising strategy for understanding campylobacteriosis in New Zealand.

  13. Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

    PubMed Central

    Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

    1999-01-01

    Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

  14. Genetic variability among Chlamydia trachomatis reference and clinical strains analyzed by pulsed-field gel electrophoresis.

    PubMed Central

    Rodriguez, P; Allardet-Servent, A; de Barbeyrac, B; Ramuz, M; Bebear, C

    1994-01-01

    Pulsed-field gel electrophoresis (PFGE) was applied to Chlamydia trachomatis reference strains representing each of the 18 serovars and to 29 clinical isolates from genital specimens collected in Bordeaux, France, or Malmö, Sweden. Comparison of the fingerprint patterns of the reference strains revealed a high level of polymorphism of the total DNA when SmaI was used (14 profiles), whereas the other enzymes, Sse8387I and ApaI, showed fewer differences. Some serovars, considered to be closely related on the basis of their antigenic determinants located on the major outer membrane protein (MOMP), such as D and Da or I and Ia, were shown to be different after PFGE of their genomic DNAs. However, serovars B and Ba and serovars L2 and L2a had identical patterns after analysis with the three endonucleases. When applied to clinical isolates, which were typed by restriction fragment length polymorphism analysis of the MOMP gene, PFGE allowed the detection of intragenotype polymorphisms and showed the identity of two strains successively isolated from the same patient. This technique seems to be an efficient tool for epidemiological studies when used in addition to serotyping or genotyping by restriction fragment length polymorphism analysis of the MOMP gene. Images PMID:7883878

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

    SciTech Connect

    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.

  16. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

    PubMed

    Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

    2016-02-01

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Multilocus Sequence Typing Compared to Pulsed-Field Gel Electrophoresis for Molecular Typing of Pseudomonas aeruginosa▿

    PubMed Central

    Johnson, Jennifer K.; Arduino, Sonia M.; Stine, O. Colin; Johnson, Judith A.; Harris, Anthony D.

    2007-01-01

    For hospital epidemiologists, determining a system of typing that is discriminatory is essential for measuring the effectiveness of infection control measures. In situations in which the incidence of resistant Pseudomonas aeruginosa is increasing, the ability to discern whether it is due to patient-to-patient transmission versus an increase in patient endogenous strains is often made on the basis of molecular typing. The present study compared the discriminatory abilities of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for 90 P. aeruginosa isolates obtained from cultures of perirectal surveillance swabs from patients in an intensive care unit. PFGE identified 85 distinct types and 76 distinct groups when similarity cutoffs of 100% and 87%, respectively, were used. By comparison, MLST identified 60 sequence types that could be clustered into 11 clonal complexes and 32 singletons. By using the Simpson index of diversity (D), PFGE had a greater discriminatory ability than MLST for P. aeruginosa isolates (D values, 0.999 versus 0.975, respectively). Thus, while MLST was better for detecting genetic relatedness, we determined that PFGE was more discriminatory than MLST for determining genetic differences in P. aeruginosa. PMID:17881548

  18. Demonstration of structural polymorphism among MB3 light chains by two-dimensional gel electrophoresis.

    PubMed

    Ishikawa, N; Kasahara, M; Ikeda, H; Ogasawara, K; Hawkin, S; Takenouchi, T; Wakisaka, A; Kikuchi, Y; Aizawa, M

    1985-01-01

    The heavy and light chain subunits of MB3 molecules were isolated from KT2 (DKT2, DR4, MB3 homozygous), ER (Dw4, DR4, MB3 homozygous), JMe (Dw5, DR5, MB3 homozygous), EBV-Sh (DSh, DRw6.2, MB3 homozygous), and EBV-Ky (DKy, DRw9, MB3 homozygous) cells and were compared with one another by two-dimensional gel electrophoresis. The MB3 light chains from KT2, ER, and EBV-Ky cells were clearly different in terms of their isoelectric points, whereas those from ER, JMe, and EBV-Sh cells were indistinguishable. No differences in charge or m.w. were noted for the MB3 heavy chains from the five cell lines. Thus, three out of the five MB3-positive, D/DR-disparate cell lines were found to express structurally distinct MB3 molecules, demonstrating that MB3 is a public serologic specificity shared by at least three structurally distinct MB (human I-A-like) molecules. Because the DR light chain subunits isolated from EBV-Wa, KT2, ER, JMe, EBV-Sh, and EBV-Ky cells differed from one another in their isoelectric points, the DR light chains were apparently more polymorphic than the MB3 light chains.

  19. Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis.

    PubMed

    Takano, E; Maki, M; Mori, H; Hatanaka, M; Marti, T; Titani, K; Kannagi, R; Ooi, T; Murachi, T

    1988-03-22

    Isolation and nucleotide sequencing of the complementary DNA for pig heart calpastatin have been completed. The amino acid sequence of 713 residues predicted from the nucleotide sequence contains five domains, each composed of approximately 140 amino acid residues. A unique N-terminal domain is followed by four mutually homologous domains. The best fit alignment of these four domains gives residue identities between any two domains of 22.5-36.0%. The analysis of the sequence similarities by several methods also suggests the existence of additional shorter repeats at intervals of 60-80 residues. The calculated molecular weight of pig calpastatin of 713 amino acid residues (Mr 77,122) is significantly lower than the value of purified pig heart calpastatin (Mr 107,000) estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The expression of the calpastatin genes in Escherichia coli and the detection of the translation products of 713, 366, and 140 amino acid residues by the specific anti-calpastatin antibody indicate that the products always migrate considerably slow on SDS-PAGE, giving an average of 1.53 for the ratio of the molecular weight estimated by SDS-PAGE to the value calculated from the amino acid sequences. It is most likely that the discrepancy in the molecular weight is caused by an anomalous behavior of calpastatin in SDS-PAGE.

  20. Motion of megabase deoxyribonucleic acid during field-inversion gel electrophoresis: Investigation by nonlocal Monte Carlo

    NASA Astrophysics Data System (ADS)

    Duke, T. A. J.; Viovy, J. L.

    1992-06-01

    We give a detailed description of a new Monte Carlo method for the simulation of the forced dynamics of long chain polymers in a constrictive environment. The model is based on the reptation theory but admits, in addition, the possibility that loops of the chain (``hernias'') may escape laterally out of the tube. A discrete representation of the molecule, in which individual chain segments are either taut or slack, permits the extensional mode of the molecule within the tube to be taken into consideration. The dynamics is modeled by the nonlocal hopping of ``defects'' (regions of slack) along the chain, with Monte Carlo rules based on the stochastic equations of motion of the taut portions of the molecule. We use the technique to investigate the motion of long deoxyribonucleic acid (DNA) molecules, containing millions of base pairs, during field-inversion gel electrophoresis. For the pulse ratios most commonly used in practice, we find that the separation patterns display two regions of band-inversion. This anomalous behavior is linked to the strong transient response of the molecules when the field is reversed; sudden field inversion induces the formation of a chain configuration shaped like an extended V after an interval of time that increases linearly with the chain length. The DNA molecules that have the minimum and the maximum migration speeds are those whose transient response times are approximately equal to the forward and the reverse pulse time, respectively.

  1. Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis.

    PubMed

    Satokari, R M; Vaughan, E E; Akkermans, A D; Saarela, M; de Vos, W M

    2001-02-01

    We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

  2. Screening for TP53 mutations in osteosarcomas using constant denaturant gel electrophoresis (CDGE).

    PubMed

    Smith-Sørensen, B; Gebhardt, M C; Kloen, P; McIntyre, J; Aguilar, F; Cerutti, P; Børresen, A L

    1993-01-01

    We have previously developed conditions to screen for TP53 point mutations inside the conserved domains II-V of the gene by using constant denaturant gel electrophoresis (CDGE). The present study reports conditions for screening more of the codons in the frequently mutated region exon 5-8 and for detecting mutations in sequences encoding functional domains in the N- and C-terminal part of the protein. The ability of the CDGE technique to detect mutations was studied using controls with known sequence deviations. The resolution power of the technique to separate different types of mutations was tested by using seven different single base pair mutants all residing in a stretch of four base pairs. All mutants were separated from the wild type. The established CDGE screening strategy was then used to look for mutations in DNA from 28 osteosarcomas. Six (21.5%) of the samples were shown to have a TP53 mutation, and the exact characterization was performed by direct sequencing. All of these were within the frequently reported mutated region exon 5-8.

  3. A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes.

    PubMed

    Ladig, Roman; Sommer, Maik S; Hahn, Alexander; Leisegang, Matthias S; Papasotiriou, Dimitrios G; Ibrahim, Mohamed; Elkehal, Rajae; Karas, Michael; Zickermann, Volker; Gutensohn, Michael; Brandt, Ulrich; Klösgen, Ralf Bernd; Schleiff, Enrico

    2011-07-01

    Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.

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

    PubMed

    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 (H2O2) and an H2O2/iron (III) chloride (FeCl3) 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.

  5. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

    PubMed Central

    Omotola, Oluwabukola B.; Heda, Rajiv P.; Avery, Jamie

    2016-01-01

    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. PMID:27582639

  6. Prokaryotic community composition revealed by denaturing gradient gel electrophoresis in the East Sea

    NASA Astrophysics Data System (ADS)

    Jang, Gwang Il; Choi, Dong Han

    2015-12-01

    To understand the temporal and spatial variation of the prokaryotic community in the East Sea, their composition was determined by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE)-sequencing techniques. The investigations were conducted twice annually in 2007 and 2009 in coastal and offshore stations. Prokaryotic abundance (PA), leucine incorporation rate, and other environmental parameters were also measured. By using the DGGE approach, we obtained 283 bacterial sequences and 160 archaeal sequences. The most frequently detected bacterial phylotypes during the investigations belonged to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes. However, their relative compositions differed in time and space. Although Alphaproteobacteria and Bacteroidetes were the dominant groups in the surface water in May 2007 and in May and October 2007, Gammaproteobacteria was dominant in mesopelagic samples. However, Gammaproteobacteria was overwhelmingly dominant in most samples in August 2009. Although Deltaproteobacteria was rarely found as a dominant bacterial group, it occupied the highest fraction in a mesopelagic sample in October 2007. Epsilonproteobacteria also showed a similar trend, although its maximal dominance was found in a mesopelagic sample in August 2009. The archaeal community was dominated overwhelmingly by members of the Euryarchaeota in most of the investigations. However, Nitrosopumilales was dominant in aphotic samples in August 2009. Further, their spatiotemporal composition at the family level changed more dynamically in the East Sea. These temporal and spatial distributions of the prokaryotic community were influenced mainly by seawater temperature and depth in the East Sea.

  7. CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis.

    PubMed

    Melani, Rafael D; Seckler, Henrique S; Skinner, Owen S; Do Vale, Luis H F; Catherman, Adam D; Havugimana, Pierre C; Valle de Sousa, Marcelo; Domont, Gilberto B; Kelleher, Neil L; Compton, Philip D

    2016-02-29

    Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

  8. Model creation of moving redox reaction boundary in agarose gel electrophoresis by traditional potassium permanganate method.

    PubMed

    Xie, Hai-Yang; Liu, Qian; Li, Jia-Hao; Fan, Liu-Yin; Cao, Cheng-Xi

    2013-02-21

    A novel moving redox reaction boundary (MRRB) model was developed for studying electrophoretic behaviors of analytes involving redox reaction on the principle of moving reaction boundary (MRB). Traditional potassium permanganate method was used to create the boundary model in agarose gel electrophoresis because of the rapid reaction rate associated with MnO(4)(-) ions and Fe(2+) ions. MRB velocity equation was proposed to describe the general functional relationship between velocity of moving redox reaction boundary (V(MRRB)) and concentration of reactant, and can be extrapolated to similar MRB techniques. Parameters affecting the redox reaction boundary were investigated in detail. Under the selected conditions, good linear relationship between boundary movement distance and time were obtained. The potential application of MRRB in electromigration redox reaction titration was performed in two different concentration levels. The precision of the V(MRRB) was studied and the relative standard deviations were below 8.1%, illustrating the good repeatability achieved in this experiment. The proposed MRRB model enriches the MRB theory and also provides a feasible realization of manual control of redox reaction process in electrophoretic analysis.

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

    PubMed

    Thomsen, Nicole; Ali, Radiya G; Ahmed, Jehangir N; Arkell, Ruth M

    2012-01-01

    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.

  10. Gel electrophoresis using a selective radical for the separation of single-walled carbon nanotubes.

    PubMed

    Mesgari, Sara; Sundramoorthy, Ashok Kumar; Loo, Leslie S; Chan-Park, Mary B

    2014-01-01

    We have applied agarose gel electrophoresis (AGE) to single-walled carbon nanotubes (SWNTs) that have been pre-reacted with metallic-selective ionic radicals and then re-suspended with sodium cholate (SC) surfactant to obtain highly purified (up to 98%) semiconducting single-walled carbon nanotubes (s-SWNTs). The proposed combination method exploits the preferential reactivity with the metallic nanotube of the radicals generated from an azo naphthalene compound (Direct Blue 71(I)) to preferentially increase the surface charge, and therefore the electrophoretic mobilities, of the metallic nanotube population under the influence of the electric field in AGE. The excellent separation achieved was verified by UV-vis-NIR and Raman spectroscopy as well as by the performance of field effect transistors fabricated with semiconducting-enriched SWNTs. FETs fabricated with -assisted AGE-separated semiconducting nanotubes exhibited mobilities of ∼3.6 to 11.7 cm(2) V(-1) s(-1) and on/off ratios from 10(2) to 10(6).

  11. On the effects of intercalators in DNA condensation: a force spectroscopy and gel electrophoresis study.

    PubMed

    Rocha, M S; Cavalcante, A G; Silva, R; Ramos, E B

    2014-05-08

    In this work we have characterized the effects of the intercalator ethidium bromide (EtBr) on the DNA condensation process by using force spectroscopy and gel electrophoresis. We have tested two condensing agents: spermine (spm(4+)), a tetravalent cationic amine which promotes cation-induced DNA condensation, and poly(ethylene glycol) (PEG), a neutral polymer which promotes DNA ψ-condensation. Two different types of experiments were performed. In the first type, bare DNA molecules disperse in solution are first treated with EtBr for intercalation, and then the condensing agent is added to the sample with the purpose of verifying the effects of the intercalator in hindering DNA condensation. In the second experiment type, the bare DNA molecules are first condensed, and then the intercalator is added to the sample in order to verify its influence on the previously condensed DNA. The results obtained with the two different experimental techniques used agree very well, indicating that previously intercalated EtBr can hinder both cation-induced and ψ-condensation, being more efficient in the first case. On the other hand, EtBr has little effect on the previously formed cation-induced condensates, but is efficient in unfolding the ψ-condensates.

  12. Application of denaturing gradient gel electrophoresis to microbial diversity analysis in Chinese Douchi.

    PubMed

    Chen, Tingtao; Jiang, Shuying; Xiong, Shunqiang; Wang, Mengjuan; Zhu, Daochen; Wei, Hua

    2012-08-15

    Douchi is a traditional Chinese soybean food which has been consumed for thousands years as an important protein source and flavouring ingredient. Studies have rarely been carried out to investigate its microbial composition and these are urgently required for the commercial labels and safety considerations. Microbial counts were statistically significant different among Douchi samples. Although the maximum diversity indexes of bacterial, bacillus and fungal polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) patterns were only 79%, 70% and 64%, some microorganisms, e.g. Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas sp., Saccharomyces cerevisiae and Pichia farinose, were found to share dominant positions in most Douchi samples. In addition, some pathogens, e.g. Staphylococcus saprophyticus, Pantoea sp., Staphylococcus sciuri, Enterobacter sp. and Staphylococcus sp., were also identified. The PCR-DGGE technique was used for the first time as an effective method to assess the microbial communities in different Chinese Douchi samples. This information may be useful in improving the product quality, reformatting production methods, extending shelf life and scaling up the fermentation process. Copyright © 2012 Society of Chemical Industry.

  13. Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis.

    PubMed

    Atanasova, Iliana; Urshev, Zoltan; Hristova, Petya; Bogatzevska, Nevena; Moncheva, Penka

    2012-01-01

    The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.

  14. [Establishment of 2-dimensional gel electrophoresis map and analysis of proteomics from human nasal polyps].

    PubMed

    He, Guang-xiang; Sun, Hong; Wang, Tian-sheng; Li, Gui; Liu, Huo-wang; Chen, Yu

    2006-08-01

    To establish 2-dimensional polyacrylamide gel electrophoresis (2-DE) map from human nasal polyps and normal nasal mucosa, and to identify differential expression proteins of 2-DE map. Samples of nasal polyps and nasal mucosa (each sample group containing 7 cases) were obtained. The total proteins were extracted and separated by immobilized pH gradient (IPG)-based 2-DE. The silver-stained 2-DE was scanned with digital Imagescanner and analyzed with ImageMaster 2-DE Elite 4.01 software. To obtain peptide mass fingerprint (PMF) of differential protein spots, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used. The PMF was searched in Swiss-Prot and TreMBL database by Pept-Ident software, to identify differential expression proteins. The well-resolved, reproducible 2-DE maps of nasal polyps and nasal mucosa were established. For the polyps tissues, the average proteins spot of three 2-DE maps was 825+/-78; and 682+/-96 spot was matched with the average matching rate of 82.7%. The average deviations of matched spot position were (1.13+/-0.16) mm in IEF direction and (1.45+/-0.21) mm in SDS-PAGE direction, respectively. For the nasal mucosa tissues, the average proteins spot of three 2-DE maps was 936+/-62; and 821+/-78 spots were matched with the average matching rate of 87.7%. After comparing the 2-DE maps of nasal polyps and nasal mucosa tissues, the protein spots were 1,458 and 1,617 respectively; and 1,026 protein spots were matched. Forty differential expression protein spots were incised from silver staining gel randomly and digested in the gel by TPCK-Trypsin. Thirty-four PMFs were obtained by MALDI-TOF-MS and 24 differential proteins were identified. The well-resolved, reproducible 2-DE maps of human nasal polyps and nasal mucosa have been successfully established. Certain differential proteins related to the pathogenesis of human nasal polyps are identified.

  15. Identification of Fur, Aconitase, and Other Proteins Expressed by Mycobacterium tuberculosis under Conditions of Low and High Concentrations of Iron by Combined Two-Dimensional Gel Electrophoresis and Mass Spectrometry

    PubMed Central

    Wong, Diane K.; Lee, Bai-Yu; Horwitz, Marcus A.; Gibson, Bradford W.

    1999-01-01

    Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 μM) and high-iron (70 μM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance. PMID:9864233

  16. Mutations of the G sup s. alpha. -subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis

    SciTech Connect

    Weinstein, L.S.; Friedman, E.; Collins, R.M.; Spiegel, A.M.; Gejman, P.V.; Kadowaki, Takashi; Gershon, E.S. )

    1990-11-01

    Affected members of most kindreds with Albright hereditary osteodystrophy have a partial deficiency of functional G{sub s}, the guanine nucleotide-binding protein that stimulates adenylyl cyclase. By use of the polymerase chain reaction to amplify genomic fragments wi