Gel Electrophoresis (gel + electrophoresis)

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Gel Electrophoresis

  • agarose gel electrophoresis
  • alkaline single cell gel electrophoresis
  • blue native polyacrylamide gel electrophoresis
  • capillary gel electrophoresis
  • cell gel electrophoresis
  • chain reaction-denaturing gradient gel electrophoresis
  • denaturing gradient gel electrophoresis
  • difference gel electrophoresis
  • dodecyl sulfate-polyacrylamide gel electrophoresis
  • dodecyl sulphate polyacrylamide gel electrophoresis
  • dodecyl sulphate-polyacrylamide gel electrophoresis
  • field gel electrophoresis
  • gradient gel electrophoresis
  • high-resolution two-dimensional gel electrophoresis
  • native gel electrophoresis
  • native polyacrylamide gel electrophoresis
  • polyacrylamide gel electrophoresis
  • polymerase chain reaction-denaturing gradient gel electrophoresis
  • pulsed field gel electrophoresis
  • pulsed-field gel electrophoresis
  • reaction-denaturing gradient gel electrophoresis
  • sds-polyacrylamide gel electrophoresis
  • single cell gel electrophoresis
  • single-cell gel electrophoresis
  • sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • sodium dodecyl sulphate polyacrylamide gel electrophoresis
  • sodium dodecyl sulphate-polyacrylamide gel electrophoresis
  • starch gel electrophoresis
  • sulfate-polyacrylamide gel electrophoresis
  • sulphate polyacrylamide gel electrophoresis
  • sulphate-polyacrylamide gel electrophoresis
  • temperature gradient gel electrophoresis
  • temporal temperature gradient gel electrophoresis
  • two-dimensional difference gel electrophoresis
  • two-dimensional gel electrophoresis
  • two-dimensional polyacrylamide gel electrophoresis

  • Terms modified by Gel Electrophoresis

  • gel electrophoresis analysis
  • gel electrophoresis pattern

  • Selected Abstracts

    Evaluation of systemic oxidative status and mononuclear leukocytes DNA damage in children with caustic esophageal stricture

    M. Kaya
    SUMMARY., Esophageal stricture (ES) due to accidentally caustic digestions is a common problem in children. Mucosal damage and repeated dilatations lead to chronic inflammation and finally ES. We investigated the oxidative status and DNA damage of children with ES. Five children with ES were compared with the same age- and sex-matched healthy subjects. Oxidative status of plasma was evaluated by measuring myeloperoxidase (MPO) activity, and total peroxide (TP) level. Anti-oxidative status of the plasma was evaluated by measuring catalase (CAT) activity, and total antioxidant response (TAR). We used the Single Cell Gel Electrophoresis (also called Comet Assay) to measure DNA strand break in peripheral blood mononuclear leukocytes. Mean MPO activity and TP levels in the ES group were significantly higher than the control group (0.83 0.35, 0.09 0.03 and 0.98 0.38, 0.34 0.20, P = 0.009 and P = 0.047 respectively). There was no significant difference in CAT activity and TAR levels between the two groups (P = 0.347). DNA damage in patients with ES was increased compared to control subjects (108.8 51.2 and 57.6 31.2 arbitrary units, respectively), but this difference was not significant statistically (P= 0.09). This study shows that systemic oxidative stress and alteration at the nuclear level occur in patients with ES, as a result of multiple dilatations and tissue injury. On the other hand, these results support that patients with ES may benefit from antioxidant treatment. [source]

    Comparison of the Electrochemical Behavior of the High Molecular Mass Cadmium Proteins in Arabidopsis thaliana and in Vegetable Plants on Using Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC-PAGE)

    ELECTROANALYSIS, Issue 1 2006
    Bernd Kastenholz
    Abstract In Arabidopsis cytosol (supernatant) and in supernatants of vegetable plants high molecular mass cadmium proteins with molecular mass 200,kDa were isolated by using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE). Because of a different electrochemical behavior of the Cd proteins in Arabidopsis and endive supernatants on using the same PAGE method, it is concluded that the high molecular mass cadmium proteins of Arabidopsis and endive possess different isoelectric points. Consequently, different chemical structures of the Cd proteins with molecular mass 200,kDa are present in Arabidopsis thaliana and in endive. During the electrophoretic separation of vegetable metalloproteins by using the Model 491 Prep Cell from BioRad, electroanalytical processes like electrode reactions may play an important role. [source]

    Temperature dependence of Fe(III) and sulfate reduction rates and its effect on growth and composition of bacterial enrichments from an acidic pit lake neutralization experiment

    GEOBIOLOGY, Issue 4 2005
    J. MEIER
    ABSTRACT Microbial Fe(III) and sulfate reduction are important electron transport processes in acidic pit lakes and stimulation by the addition of organic substrates is a strategy to remove acidity, iron and sulfate. This principle was applied in a pilot-scale enclosure in pit lake 111 (Brandenburg, Germany). Because seasonal and spatial variation of temperature may affect the performance of in situ experiments considerably, the influence of temperature on Fe(III) and sulfate reduction was investigated in surface sediments from the enclosure in the range of 4,28 C. Potential Fe(III) reduction and sulfate reduction rates increased exponentially with temperature, and the effect was quantified in terms of the apparent activation energy Ea measuring 42,46 kJ mol,1 and 52 kJ mol,1, respectively. Relatively high respiration rates at 4 C and relatively low Q10 values (,2) indicated that microbial communities were well adapted to low temperatures. In order to evaluate the effect of temperature on growth and enrichment of iron and sulfate-reducing bacterial populations, MPN (Most Probable Number) dilution series were performed in media selecting for the different bacterial groups. While the temperature response of specific growth rates of acidophilic iron reducers showed mesophilic characteristics, the relatively high specific growth rates of sulfate reducers at the lowest incubation temperature indicated the presence of moderate psychrophilic bacteria. In contrast, the low cell numbers and low specific growth rates of neutrophilic iron reducers obtained in dilution cultures suggest that these populations play a less significant role in Fe and S cycling in these sediments. SSCP (Single-Strand Conformation Polymorphism) or DGGE (Denaturing Gradient Gel Electrophoresis) fingerprinting based on 16S rRNA genes of Bacteria indicated different bacterial populations in the MPN dilution series exhibiting different temperature ranges for growth. [source]

    Hypoxia-like effect of Cobalt Chromium alloy micro particles on fibroblasts in vitro

    Bernadette K. Madathil
    Abstract Periprosthetic osteolysis leading to asceptic loosening remains the primary cause of failure of joint replacement. Although many inflammatory cell types have been implicated, the exact pathomechanisms of asceptic loosening have not been delineated. In the present study we have adopted a proteomic approach to elucidate the initial signals that are expressed to particulate material, using an in vitro cell culture system. Human lung fibroblasts MRC-5 were cultured with Cobalt Chromium (CoCr ASTM F-75, 1,7,m) particles. Cells were harvested after 72,h incubation and total cellular proteins extracted for downstream analysis via 2D Gel Electrophoresis and tandem mass spectrometry using MALDI-TOF-TOF-MS. Thirteen protein spots showed greater than twofold increase, following 72,h incubation of fibroblast with CoCr particles. Four of these proteins were identified by tandem mass spectrometry. These were Annexin II, Pyruvate kinase, Triose phosphate isomerase, and N-myc downstream regulated gene 1 protein. Cobalt is a hypoxia mimicking agent and N-myc downstream regulated gene 1 protein, Triose phosphate isomerase, Pyruvate kinase, and Annexin II are important hypoxia regulated gene products that are found to be over expressed in cellular oxidative stress response. Our data indicates that exposure of fibroblast to CoCr alloy induces the transition of these cells into a hypoxia like state and oxidative stress even in normoxic culture conditions. The study reflects the possibility of the presence of a hypoxic environment in the periprosthetic tissue surrounding metallic implants. Published by Wiley Periodicals, Inc. J Orthop Res 28:1360,1367, 2010 [source]

    Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

    R.S. Peixoto
    Aim: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. Methods: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. Results: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. Conclusions, Significance and Impact of the Study: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE. [source]

    An Update on Data Standards for Gel Electrophoresis

    Andrew R Jones Dr.
    The use of gel electrophoresis to separate and, in some instances, to quantify the abundance of large numbers of proteins from complex mixtures, has been well established for several decades. The quantity of publicly available data is still relatively modest due to a lack of community accepted data standards, tools to facilitate the data sharing process and controlled vocabularies to ensure that consistent terminology is used to describe the experimental methodology. It is becoming widely recognised that there are significant benefits in data sharing for proteomics, allowing results to be verified and new findings to be generated by re-analysis of published studies. We report on standards development by the Gel Analysis Workgroup of the Proteomics Standards Initiative. The workgroup develops reporting requirements, data formats and controlled vocabularies for experimental gel electrophoresis, and informatics performed on gel images. We present a tutorial on how such resources can be used and how the community should get involved with the on-going projects. Finally, we present a roadmap for future developments in this area. [source]

    Effect of dietary administration of probiotics on growth and intestine functionality of juvenile Senegalese sole (Solea senegalensis, Kaup 1858)

    Abstract The effects of the dietary administration of two bacterial probiotic strains (Ppd11 and Pdp13) from the Alteromonadaceae family for 60 days, were assessed by measuring growth and feed efficiency, activities of leucine aminopeptidase and alkaline phosphatase and structural changes in the intestine of juvenile Senegalese sole. In addition, the profile of intestinal microbiota was studied by Denaturing Gradient Gel Electrophoresis. Growth and nutrient utilization were significantly higher in fish receiving probiotics than in those fed the control diet. No differences were observed in proximal composition between treatments, though higher lipid muscle content was measured in fish receiving Pdp13. Those fish also exhibited higher activities of AP when compared to Ppd11 and control groups. The profile of intestinal microbiota clearly separated those fish receiving probiotics from those of the control group. Microscopical examination revealed accumulation of lipid droplets in the enterocytes of fish receiving the control diet, but not in those fed on probiotics. Interactions between those structural changes and growth performance are discussed. [source]

    Biotec Visions July 2009

    Article first published online: 17 JUL 200
    News: Mutagenic biodiesel blends , Technicolor cancer imaging , Anticancer nanoparticle , Increased oxygen transfer in baffled microtiter plates , Transgenic barley growing on acid soil , Brain music , Laser light-induced brain waves , First genome sequence of ruminant species Special issues: Cytometry of microbes , Food-borne Mycotoxins Book highlights: Biotech funding trends , Biotechnology in Flavor Production Opinion: Another biofuel blunder? Tips and tricks: Good to know: Gel Electrophoresis Test your knowledge. Do you recognize this? Most read Writing tips Briefs: A hypothetical new model of LDL , Gaden Award , Patenting hES cells in Europe [source]

    Microfabricated Polymer Chip for Capillary Gel Electrophoresis

    Jong Wook Hong
    A polymer (PDMS: poly(dimethylsiloxane)) microchip for capillary gel electrophoresis that can separate different sizes of DNA molecules in a small experimental scale is presented. This microchip can be easily produced by a simple PDMS molding method against a microfabricated master without the use of elaborate bonding processes. This PDMS microchip could be used as a single use device unlike conventional microchips made of glass, quartz or silicon. The capillary channel on the chip was partially filled with agarose gel that can enhance separation resolution of different sizes of DNA molecules and can shorten the channel length required for the separation of the sample compared to capillary electrophoresis in free-flow or polymer solution format. We discuss the optimal conditions for the gel preparation that could be used in the microchannel. DNA molecules were successfully driven by an electric field and separated to form bands in the range of 100 bp to 1 kbp in a 2.0% agarose-filled microchannel with 8 mm of effective separation length. [source]

    Stabilization of PbS Nanocrystals by Bovine Serum Albumin in its Native and Denatured States

    Mandeep Singh Bakshi
    Abstract PbS nanocrystals (NCs) are synthesized in aqueous phase within a temperature range of 40,80,C in the presence of native and denatured states of bovine serum albumen (BSA) as the capping/stabilizing agent. The NCs are characterized with the help of field-emission scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, and energy-dispersive X-ray analysis. At 40,C, large ball-shaped NCs (145,,37,nm) with small surface protrusions are formed when 1,,10,4,g mL,1 BSA is used. As the reaction temperature is increased towards 80,C, the size of NCs decreases and they acquire somewhat cubic geometries (49.1,,7.0,nm) due to a change in the capping behavior of BSA between its native and denatured states. The native and denatured states of BSA are simultaneously studied by fluorescence spectroscopy using tryptophan emission, and pH measurements with respect to time and temperature. Gel electrophoresis is used to determine the polarity of the BSA capped NCs. Only the small sized NCs conjugated with relatively larger amounts of BSA show a displacement towards the positively charged electrode in comparison to larger NCs with lower amounts of BSA capping. It was concluded that the denatured state of BSA is more effective in controlling the crystal growth of PbS than its native state especially in the low concentration range. [source]

    Fabrication, characterization and in vitro evaluation of poly(D,L -lactide- co -glycolide) microparticles loaded with polyamidoamine,plasmid DNA dendriplexes for applications in nonviral gene delivery

    Janjira Intra
    Abstract We report, for the first time, on the preparation, characterization and in vitro testing of poly(D,L -lactide- co -glycolide) (PLGA) microparticles loaded with polyamidoamine (PAMAM),plasmid DNA (pDNA) dendriplexes. Loading of pDNA into the PLGA microparticles increased by 150% when pDNA was first complexed with PAMAM dendrimers relative to loading of pDNA alone. Scanning electron microscopy (SEM) showed that the presence of PAMAM dendrimers in the PLGA microparticles created porous features and indentations on the surface of the microparticles. Loading PLGA microparticles with PAMAM,pDNA dendriplexes lowered the average PLGA microparticle size and changed the surface charge of the microparticles from negative to positive when compared to PLGA microparticles loaded with pDNA alone. The zetapotential and buffering capacity of the microparticles increased as the generation of the PAMAM dendrimer loaded in the PLGA microparticles increased. Gel electrophoresis assays showed that all the PLGA microparticle formulations were able to entrap the pDNA within the PLGA matrix. There was no significant difference in the cytotoxicity of PLGA microparticles loaded with PAMAM,pDNA dendriplexes when compared to PLGA microparticles loaded with pDNA alone. Furthermore, and in contrast to PAMAM dendrimers alone, the generation of the PAMAM dendrimer loaded in the PLGA microparticles had no significant impact on cytotoxicity or transfection efficiencies in human embryonic kidney (HEK293) or Monkey African green kidney fibroblast-like (COS7) cells. The transfection efficiency of PLGA microparticles loaded with generation 3 (G3) PAMAM,pDNA dendriplexes was significantly higher than PLGA microparticles loaded with pDNA alone in HEK293 and COS7 cells. PLGA microparticles loaded with G3 PAMAM,pDNA dendriplexes generated equivalent transfection efficiencies as (G3 to G6) PAMAM,pDNA dendriplexes alone in COS7 cells when the transfection was carried out in serum containing media. The delivery system developed in this report has low toxicity, high pDNA loading efficiencies and high transfection efficiencies that are not reduced in the presence of serum. A delivery system with these characteristics is expected to have significant potential for translational applications. 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:368,384, 2010 [source]

    How do helix,helix interactions help determine the folds of membrane proteins?

    PROTEIN SCIENCE, Issue 4 2003
    Perspectives from the study of homo-oligomeric helical bundles
    FRET, fluorescence resonance energy transfer; NBD, 7-nitrobenz-2-oxa-1,3-diazole; C-14 betaine, N -tetradecyl- N,N -dimethyl-3-ammonio-1-propanesulfonate; MF, mole fraction Abstract The final, structure-determining step in the folding of membrane proteins involves the coalescence of preformed transmembrane helices to form the native tertiary structure. Here, we review recent studies on small peptide and protein systems that are providing quantitative data on the interactions that drive this process. Gel electrophoresis, analytical ultracentrifugation, and fluorescence resonance energy transfer (FRET) are useful methods for examining the assembly of homo-oligomeric transmembrane helical proteins. These methods have been used to study the assembly of the M2 proton channel from influenza A virus, glycophorin, phospholamban, and several designed membrane proteins,all of which have a single transmembrane helix that is sufficient for association into a transmembrane helical bundle. These systems are being studied to determine the relative thermodynamic contributions of van der Waals interactions, conformational entropy, and polar interactions in the stabilization of membrane proteins. Although the database of thermodynamic information is not yet large, a few generalities are beginning to emerge concerning the energetic differences between membrane and water-soluble proteins: the packing of apolar side chains in the interior of helical membrane proteins plays a smaller, but nevertheless significant, role in stabilizing their structure. Polar, hydrogen-bonded interactions occur less frequently, but, nevertheless, they often provide a strong driving force for folding helix,helix pairs in membrane proteins. These studies are laying the groundwork for the design of sequence motifs that dictate the association of membrane helices. [source]

    Structure, DNA Binding Studies and Cytotoxicity of Complex [Pd(phen)(L -asp)]3H2O

    Enjun GAO
    Abstract The palladium(II) complex of [Pd(phen)(L -asp)]3H2O (phen=1,10-phenanthroline, H2L-asp=L -aspartic acid) has been synthesized from a solution reaction and analyzed by elemental analyses, 1H NMR and IR spectra. Moreover, the complex has been structurally characterized by single-crystal X-ray diffractometry. The cytotoxicity assay of the complex and cis -DDP as reference substance against three different cancer cell lines (Hela, Hep-G2 and KB) has been conducted. The results show that the Pd complex exhibits higher cytotoxicity against Hela system. The study on the interaction of the Pd complex with fish sperm DNA (FS-DNA) has been performed with diverse spectroscopic techniques, showing that the complex is bound to the fish sperm DNA via an intercalative mode. Gel electrophoresis assay demonstrates the ability of the complex to cleave the pBR 322 plasmid DNA. [source]

    Interleukin-1 receptor antagonist and tumour necrosis factor-alpha gene polymorphisms in Turkish patients with allergic contact dermatitis

    CONTACT DERMATITIS, Issue 2 2009
    Ilgen Ertam
    Background: It has been shown that the family of interleukin-1 receptor antagonist (IL-1 RA) and tumour necrosis factor-alpha (TNF,) genes are polymorphic and related to some inflammatory diseases. Allergic contact dermatitis is the classic presentation of delayed-type hypersensitivity responses to exogenous agents. A number of genes playing role in inflammatory response may be associated with allergic contact dermatitis. Objectives: To investigate whether there is an association between IL-1RA and TNF, gene polymorphisms and allergic contact dermatitis in Turkish patients with allergic contact dermatitis. Methods: This study was performed by the collaboration of Departments of Dermatology and Medical Genetics, Ege University, Faculty of Medicine. A total of 50 patients (31 females and 19 males) with allergic contact dermatitis, and 100 age- and sex-matched controls (58 females and 42 males) were included in the study. IL-1RA Variable Number of Tandem Repeats (VNTR) polymorphism in intron 2 and TNF,-308G-A polymorphism were genotyped by using polymerase chain reaction and agarose gel electrophoresis. Results: The frequency of IL-1RA 1/2 (48%) genotype was significantly higher (P = 0.002) in patient group than that is found in control group (22%). The frequency of TNF, (TNF G-308A) G/G genotype was significantly higher in patient group (68%) than that is found in control group (31%) (P = 0.008). Conclusions: Our findings suggest that TNF, (G/G) gene polymorphism may play role in susceptibility to allergic contact dermatitis in Turkish patients. [source]

    Control of flatfish sperm motility by CO2 and carbonic anhydrase

    CYTOSKELETON, Issue 3 2003
    Kazuo Inaba
    Abstract Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25,100 mM HCO3,, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3,. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3, -arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/ HCO3, -dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm. Cell Motil. Cytoskeleton 55:174,187, 2003. 2003 Wiley-Liss, Inc. [source]

    Effect of celecoxib on cyclooxygenase-2 expression and possible variants in a patient with Barrett's esophagus

    G. A. Jacobson
    SUMMARY., Cyclooxygenase-2 (COX-2) expression is increased in metaplastic and dysplastic Barrett's esophageal epithelium and it is thought that selective COX-2 inhibitors could offer hope as chemoprevention therapy. The aim of the study was to investigate the in vivo effect of celecoxib on COX-2 expression in patients with Barrett's esophagus and no recent history of non-steroidal anti-inflammatory drug use. Endoscopic mucosal biopsy specimens were collected at baseline and after 28 days of therapy in a patient treated with celecoxib 200 mg twice daily. Samples were analyzed for COX-2 expression by immunoblot analysis with chemiluminescence detection. COX-2 expression was found to decline 20% and 44% at two different biopsy sites compared to the baseline sample. Longer exposures revealed a number of previously unidentified proteins above and below the 67 kDa COX-2 protein including 38 kDa and 45 kDa proteins which were present only at study completion consistent with up-regulation after celecoxib therapy. Further investigations of the 38 kDa and 45 kDa proteins were undertaken using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with immunoblot and MALDI-TOF (matrix assisted laser desorption ionization , time of flight) analysis but no matches were found and results were inconclusive. Unmatched masses from MALDI-TOF peptide mass fingerprinting were compared with human COX-2 (67 kDa) and COX-2b (39 kDa) using unspecific cleavage. Peptide sequence homology with COX-2 and COX-2b was found for a length of 19 amino acids. Based on immunodetection, molecular weight and equivical MALDI-TOF results, one of these up-regulated proteins may be COX-2b. [source]

    Comparison of the Electrochemical Behavior of the High Molecular Mass Cadmium Proteins in Arabidopsis thaliana and in Vegetable Plants on Using Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC-PAGE)

    ELECTROANALYSIS, Issue 1 2006
    Bernd Kastenholz
    Abstract In Arabidopsis cytosol (supernatant) and in supernatants of vegetable plants high molecular mass cadmium proteins with molecular mass 200,kDa were isolated by using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE). Because of a different electrochemical behavior of the Cd proteins in Arabidopsis and endive supernatants on using the same PAGE method, it is concluded that the high molecular mass cadmium proteins of Arabidopsis and endive possess different isoelectric points. Consequently, different chemical structures of the Cd proteins with molecular mass 200,kDa are present in Arabidopsis thaliana and in endive. During the electrophoretic separation of vegetable metalloproteins by using the Model 491 Prep Cell from BioRad, electroanalytical processes like electrode reactions may play an important role. [source]

    Simultaneous detection of genetically modified organisms by multiplex ligation-dependent genome amplification and capillary gel electrophoresis with laser-induced fluorescence

    ELECTROPHORESIS, Issue 13 2010
    Virginia Garca-Caas
    Abstract In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis. [source]

    Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampus

    ELECTROPHORESIS, Issue 11 2010
    Seok Heo
    Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. Modiro v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source]

    Cover Picture: Electrophoresis 10'2010

    ELECTROPHORESIS, Issue 10 2010
    Article first published online: 18 MAY 2010
    Issue no. 10 is a regular issue comprising 19 manuscripts distributed over four distinct parts. Part I is on microfluidics and miniaturized systems and has 5 articles; Part II is on nucleic acids with 4 articles on restriction endonuclease fingerprinting, mutation detection and DNA separation and detection; Part III has 7 articles on monolithic stationary phases for CEC, single walled carbon nanohorns as pseudo-stationary phases for CEC and EKC, MEEKC, cyclodextrin-modified gold nanoparticles for enantioseparations by CEC, use of divalent dipeptides as counter ions in CE and capillary coating for CE of proteins; and Part IV has 3 articles on proteomics methodologies. Featured articles include: Microfluidic preparative free-flow isoelectric focusing in a triangular channel: System development and characterization ((10.1002/elps.200900577)) Separation and recovery of nucleic acids with improved biological activity by acid-degradable polyacrylamide gel electrophoresis ((10.1002/elps.200900783)) Evaluation of the performance of single-walled carbon nanohorns in capillary electrophoresis ((10.1002/elps.200900628)) The inter- and intra-operator variability in manual spot segmentation and its effect on spot quantitation in two dimensional electrophoresis analysis. ((10.1002/elps.200900674)) [source]

    Cover Picture: Electrophoresis 3'2010

    ELECTROPHORESIS, Issue 3 2010
    Article first published online: 29 JAN 2010
    Issue no. 3 is a regular issue with Emphasis on "Proteins and Proteomics". The first part has 8 articles on proteins and proteomics covering various topics, e.g. preparative divergent flow IEF, multichannel gel electrophoresis, capillary gel electrophoresis, nanoparticle-based CE of proteins, 2-DE in a radial gel format, depletion of high abundance proteins, and proteomic investigation of fetal brain and lentil seed. The remaining 10 articles are concerned with nucleic acids, gene expression, methodologies and application. Featured articles include: Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples ((10.1002/elps.200900484)) SDS-PAGE and two-dimensional maps in a radial gel format ((10.1002/elps.200900526)) Analysis of Effect of Electrolyte Types on Electrokinetic Energy Conversion in Nanoscale Capillaries ((10.1002/elps.200900409)) A simple method to determine the surface charge in microfluidic channels ((10.1002/elps.200900603)) [source]

    Determination of DNA methylation by COBRA: A comparative study of CGE with LIF detection and conventional gel electrophoresis

    ELECTROPHORESIS, Issue 17 2009
    Simon Goedecke
    Abstract 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. [source]

    Cover Picture: Electrophoresis 14'09

    ELECTROPHORESIS, Issue 14 2009
    Article first published online: 28 JUL 200
    Issue no. 14 is an Emphasis Issue with 9 articles on various aspects of "Proteins and Proteomics" while the remaining 14 articles are arranged into 4 different parts including "Microfluidics and Miniaturization", "Genotyping and Transcriptomics", "Enantioseparations", and "Nanoparticles and Abused Drugs Analyses". Selected articles are: Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresis ((10.1002/elps.200900026)) 2-D difference in gel electrophoresis combined with Pro-Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets ((10.1002/elps.200800780)) Microvalves actuated sandwich immunoassay on an integrated microfluidic system ((10.1002/elps.200800818)) Chemical gradient-mediated melting curve analysis for genotyping of SNPs ((10.1002/elps.200800729)) [source]

    Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresis

    ELECTROPHORESIS, Issue 14 2009
    Jingdan Liang
    Abstract Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps. [source]

    Cover Picture: Electrophoresis 3'09

    ELECTROPHORESIS, Issue 3 2009
    Article first published online: 11 FEB 200
    This is a regular issue with an emphasis on "Fundamentals Methodologies and Instrumentation" assembling 11 articles in various research areas on fundamentals, methods development, instrumental design, detection and sensitivity enhancement approaches. The remaining articles are on proteins and proteomics analyses by various electrophoretic approaches. Selected topics of issue 3 are: Capillary Electrophoresis-based detection of Methicillin-resistant Staphylococcus aureus (MRSA CE-based detection of methicillin-resistant Staphylococcus aureus A portable capillary electropherograph equipped with a cross-sampler and a contactless-conductivity detector for the detection of the degradation products of chemical warfare agents in soil extracts Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes [source]

    Errata: Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes

    ELECTROPHORESIS, Issue 24 2008
    Frank Krause Dr.
    No abstract is available for this article. [source]

    Quantification of SMN1 and SMN2 genes by capillary electrophoresis for diagnosis of spinal muscular atrophy

    ELECTROPHORESIS, Issue 13 2008
    Chun-Chi Wang
    Abstract We present the first CE method for the separation and quantification of SMN1 and SMN2 genes. Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder deleted or mutated in SMN1 gene and retained at least one copy of SMN2 gene. However, these two genes are highly homologous, differentiation and quantification of SMN1 and SMN2 are therefore required in diagnosis to identify SMA patients and carriers. We developed a fluorescence-labeled conformation-sensitive CE method to quantitatively analyze PCR products covering the variable position in the SMN1/SMN2 genes using a copolymer solution composed of hydroxyethylcellulose and hydroxypropylcellulose. The DNA samples included 24 SMA patients, 52 parents of SMA patients (obligatory carriers), and 255 controls. Those 331 samples were blind analyzed to evaluate the method, and the results compared with those obtained using denaturing HPLC (DHPLC). Validation of accuracy was performed by comparing the results with those of DHPLC. Nine of total samples showed different results. Diagnosis of one fetus DNA among them was related to abortion or not, which was further confirmed by gel electrophoresis and DNA sequencing. Our method showed good coincidence with them, and proved the misdiagnosis of DHPLC. This simple and reliable CE method is a powerful tool for clinical genotyping of large populations to detect carriers and SMA patients. [source]

    A novel approach for analysis of oligonucleotide,cisplatin interactions by continuous elution gel electrophoresis coupled to isotope dilution inductively coupled plasma mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry

    ELECTROPHORESIS, Issue 7 2008
    Wolfram Brchert
    Abstract In this work we present a novel approach for in vitro studies of cisplatin interactions with 8-mer oligonucleotides. The approach is based on the recently developed coupling of continuous elution gel electrophoresis (GE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) with the aim of monitoring the interaction process between this cytostatic drug and the nucleotides. In contrast to existing methods, the electrophoretic separation conditions used here allow both the determination of the reaction kinetics in more detail as well as the observation of dominant intermediates. Two different nucleotides sequences have been investigated for comparison purposes, one containing two adjacent guanines (5,-TCCGGTCC-3,) and one with a combination of thymine and guanine (5,-TCCTGTCC-3,), respectively. In order to gain further structural information, MALDI-TOF MS measurements have been performed after fraction collection. This allows for identification of the intermediates and the final products and confirms the stepwise coordination of cisplatin via monoadduct to bisadduct formation. Furthermore, the ICP-MS results were quantitatively evaluated in order to calculate the kinetics of the entire process. [source]

    Cover Picture: Electrophoresis 6/2008

    ELECTROPHORESIS, Issue 6 2008
    Article first published online: 18 MAR 200
    Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. In addition, issue no. 6 features a series of 7 important papers on "Microfluidics and Miniaturization" dealing with LCD-based optoelectronic tweezers, cell sorting, single cell clone analysis and cultivation, integrated ITP stacking and gel electrophoresis, floating injection, electrokinetic flows on thermosensitive surfaces and flow velocity measurement in CE chip instruments. [source]

    Diversity of rice glutelin polypeptides in wild species assessed by the higher-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subunit-specific antibodies

    ELECTROPHORESIS, Issue 6 2008
    Nadar Khan
    Abstract In efforts to find genetic resources with high nutritional value of rice seed, we assessed the diversity of the major storage protein glutelin in 13 wild and 2 cultivated rice species by a unique SDS-PAGE method and subunit-specific antibodies. Maximum separation of microheterogeneous glutelin ,-polypeptides, which is a prerequisite for the diversity evaluation, could be attained by SDS-PAGE performed at higher temperature (45C) than the generally employed temperatures (4,25C). Seven antipeptide antibodies were raised against subunit-specific epitope sequences designed at five sites from four variable regions spanning the glutelin ,-polypeptides. High specificity of each antibody was confirmed using rice glutelin mutants, and demonstrated considerable variation in amino acid sequence and accumulation level of glutelin subunit in wild species, in combination with the higher-temperature SDS-PAGE. The degree of the variation was, however, changed according to the site of variable regions and the type of subunit. Some wild species accumulated nutritious GluB subunits more than cultivated rice. The wild species Oryza longiglumis and O. brachyantha had glutelin with low reactivity against most antibodies examined in this study, reflecting the significant divergence. Such wild species may hopefully serve as important genetic resources for nutritional improvement of cultivated rice. [source]