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Gel electrophoresis

About: Gel electrophoresis is a research topic. Over the lifetime, 26026 publications have been published within this topic receiving 1113565 citations.


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Journal ArticleDOI
TL;DR: White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile.
Abstract: White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 + 6) WSSV structural protein genes. Only 61.5% of the WSSV structural genes have a polyadenylation signal, and preliminary analysis by 3' rapid amplification of cDNA ends suggested that some structural protein genes produced mRNA without a poly(A) tail. Microarray analysis showed that gene expression started at 2, 6, 8, 12, 18, 24, and 36 hpi for 7, 1, 4, 12, 9, 5, and 1 of the genes, respectively. Based on similarities in their time course expression patterns, a clustering algorithm was used to group the WSSV structural genes into four clusters. Genes that putatively had common or similar roles in the viral infection cycle tended to appear in the same cluster.

241 citations

Journal ArticleDOI
TL;DR: When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis, in contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS).
Abstract: SUMMARY: When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immuno-electrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29000, while B had a molecular weight of 190000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.

241 citations

Journal ArticleDOI
TL;DR: The results suggest that the 50-Kd phosphoprotein may be an autophosphorylatable subunit of the Synapsin I Kinase or may exist in a complex with it.
Abstract: A calcium/calmodulin-dependent protein kinase, which phosphorylates a synaptic vesicle-associated protein designated Synapsin I, has been shown to be present in both soluble and particulate fractions of rat brain homogenates. In the present study, the particulate activity was solubilized by washing with a low ionic strength solution, and the enzymes from the two fractions were partially purified by ion exchange chromatography and calmodulin-Sepharose affinity chromatography. By each of several criteria, the partially purified enzymes from the two sources were indistinguishable. These criteria included specificity for various substrate proteins, concentration dependence of activation by calcium and calmodulin, pH dependence, and apparent affinities for the substrates Synapsin I and ATP. The mild conditions that released the particulate enzyme indicated that it was not tightly bound to the membrane and suggested that it may exist in a dynamic equilibrium between soluble and particulate-bound states. The partially purified enzyme preparations from both the soluble and particulate fractions contained three proteins that were phosphorylated in the presence of calcium and calmodulin, a 50-kilodalton (Kd) protein and two proteins in the 60-Kd region. When compared by phosphopeptide mapping and two-dimensional gel electrophoresis, the proteins were indistinguishable from three proteins of corresponding molecular weights that were shown by Schulman and Greengard (Schulman, H., and P. Greengard (1978) Nature 271: 478-479) to be prominent substrates for calcium/calmodulin-dependent protein kinase in a crude particulate preparation from rat brain. The 50-Kd substrate was the major Coomassie blue staining protein in both partially purified enzyme preparations. The peak of this protein coincided with that of enzyme activity during DEAE-cellulose and calmodulin-Sepharose chromatography. These results suggest that the 50-Kd phosphoprotein may be an autophosphorylatable subunit of the Synapsin I Kinase or may exist in a complex with it.

241 citations

Journal ArticleDOI
TL;DR: Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing and recognized not only native but also partially deglycosylated protein.
Abstract: Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.

241 citations

Journal Article
TL;DR: A new method for the detection of high molecular weight DNA fragments on conventional agarose gels is presented, together with a rationale for the analysis of DNA fragmentation during apoptosis.
Abstract: DNA fragmentation during apoptosis proceeds through an ordered series of stages commencing with the production of DNA fragments of 300 kbp, which are then degraded to fragments of 50 kbp. The 50-kbp fragments are further degraded, in some but not all cells, to smaller fragments (10-40 kbp) and release the small oligonucleosome fragments that are recognized as the characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of the initial stages of DNA fragmentation using pulsed-field gel electrophoresis or a combination of pulsed-field gel electrophoresis and conventional agarose gel electrophoresis that allows detection of the DNA ladder in the same sample. A new method for the detection of high molecular weight DNA fragments on conventional agarose gels is presented, together with a rationale for the analysis of DNA fragmentation during apoptosis.

240 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202364
2022116
2021108
2020104
2019120
2018147