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Dodecyl sulphate/polyacrylamide-gel electrophoresis at low pH values and low temperatures.

ABSTRACT: A simple method is described for dodecyl sulphate/polyacrylamide-gel electrophoresis of pH- and temperature-labile biological intermediates. The method is based on a catalyst system that works at temperatures of 2--4 degrees C and pH values of 2--4 and an appropriate buffer system containing Li+ or Tris [CH2OH--C(CH2OH)2--NH3+] instead of Na+. This system does not lead to the precipitation of 1% dodecyl sulphate.

SUBMITTER: Lichtner R

PROVIDER: S-EPMC1161216 | biostudies-other | 1979 Sep

REPOSITORIES: biostudies-other

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Highly purified agarose as stacking gel in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Project description:A modified sodium dodecyl sulphage/polyacrylamide-gel-electrophoretic method is described that utilizes highly purified agarose as stacking gel. The same electrophoretic resolution of different marker proteins is found as when polyacrylamide is used as stacking gel, but the background staining seen when polyacrylamide is used as stacking gel is decreased.

| S-EPMC1161930 | biostudies-other

Anomalous migration of leghaemoglobin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Project description:The estimate of the molecular weight of leghaemoglobin by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is about 20% too low. This is due to an anomalously high limiting relative mobility. Leghaemoglobin binds 1.4 g of sodium dodecyl sulphate/g of protein with a concomitant decrease in the helical content from 71-72% to 49-51%.

| S-EPMC1184216 | biostudies-other

A method for the preparation of low-pH dodecyl sulphate/polyacrylamide-gradient gels.

Project description:1. A low-pH lithium dodecyl sulphate/polyacrylamide-gradient slab-gel system, suitable for electrophoresis, is described, and the migration properties of standard proteins are compared on this and conventional high-pH gels. 2. Cytochrome oxidase may be partially resolved into its component polypeptides. The order of migration of these is, however, dependent on the pH of the gel system.

| S-EPMC1162698 | biostudies-other

Differentiation of Bartonella species by a microimmunofluorescence assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblotting.

Project description:Bartonella species can be differentiated by microimmunofluorescence assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with murine polyclonal antisera to Bartonella henselae, B. quintana, B. elizabethae, and B. bacilliformis. A pairwise comparison on the basis of SDS-PAGE protein profiles demonstrated similarity values for proteins of different Bartonella species ranging from 28.6 to 86.4%. Antigenic relationships revealed by immunoblotting with murine antisera were equivalent to those of proteins observed by SDS-PAGE. A dendrogram obtained on the basis of protein bands of SDS-polyacrylamide gels showed that Bartonella species could be divided into three groups. B. bacilliformis was distinct from all other Bartonella species; B. grahamii, B. taylorii, B. doshiae, and B. vinsonii formed a cluster, as did B. henselae, B. quintana, B. elizabethae, and B. clarridgeiae. These relationships were consistent with those revealed by parsimony trees derived from 16S rRNA and gltA gene sequencing. SDS-PAGE analysis showed that 120-, 104-, 85-, 71-, 54-, 47-, 40-, 33-, 30-, and 19-kDa proteins were present in all species, with the 54-kDa protein being the most dominant. Proteins with a molecular mass of less than 54 kDa allow the differentiation of species and are a possible target for future species-specific antibodies and antigens.

| S-EPMC95924 | biostudies-literature

Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Project description:Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). A method is presented for the rapid purification of InsP3 3-kinase from bovine brain by calmodulin (CaM)-Sepharose affinity chromatography. Maximal activation of the purified InsP3 3-kinase by Ca2+/CaM was 6-7-fold as compared with the activity measured in the presence of EGTA (1 mM) and 10 microM-InsP3. At 10 microM-InsP3 and 0.1 mM free Ca2+, half-maximal activation required about 2 nM-CaM. The mechanism of activation by CaM appeared to be an increase in the maximal velocity of the enzyme without a substantial change in the Km for InsP3. Further purification was achieved by phosphocellulose chromatography eluted with ATP. Specific activity of the purified enzyme at 37 degrees C and 10 microM-InsP3 was 10-20 mumol/min per mg. The apparent Mr of the enzyme, determined by f.p.l.c.-gel filtration, was estimated as about 44,000. The purified InsP3 3-kinase was subjected to SDS/10%-polyacrylamide-gel electrophoresis. InsP3 3-kinase activity was associated with three silver-stained bands, which migrated with apparent Mr values of approx. 52,000, 38,000 and 35,000.

| S-EPMC1138851 | biostudies-other

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

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| S-EPMC3755479 | biostudies-literature

A versatile polyacrylamide gel electrophoresis based sulfotransferase assay.

Project description:BackgroundSulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes.ResultsA universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. This assay has been successfully applied to substrates as small as alpha-naphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of +/- 0.2 of the actual relative mobilities.ConclusionThe versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.

| S-EPMC2834601 | biostudies-literature

Use of PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques for differentiation of Prevotella intermedia sensu stricto and Prevotella nigrescens.

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| S-EPMC88649 | biostudies-literature

The identification of rat intestinal membrane enzymes after electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate.

Project description:Brush-border membranes were isolated from the rat small intestine and then treated with sodium dodecyl sulphate under non-reducing conditions at room temperature. Analysis of the solubilized components by polyacrylamide-gel electrophoresis identified three major glycoproteins that co-migrate with glucoamylase-maltase-sucrase, lactase and isomaltase-maltase-sucrase activities. High activities of alkaline phosphatase and trehalase were detectable, but they could not be attributed to distinct co-migrating protein bands. Analysis of mucosa from the distal small intestine by the same methods showed a pattern of bands different from that obtained with the proximal intestine, which appeared to correlate with the relative deficiency of some of the enzymes in the distal region.

| S-EPMC1185885 | biostudies-other

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