Gel electrophoresis

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

Phosphorylation of Smooth Muscle Myosin Light Chain Kinase by the Catalytic Subunit of Adenosine 3': 5'-Monophosphate-dependent

Abstract: SUMMARY Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator coupled to Sepharose and found to have a molecular weight of 125,000 + 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a a-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.

Resolution of histones by polyacrylamide gel electrophoresis in presence of nonionic detergents.

Abstract: Publisher Summary This chapter describes the technique for the resolution of histones polyacrylamide gel electrophoresis in presence of nonionic detergents. Histones are difficult to resolve by classical biochemical fractionation techniques because of their similarity in size and charge, their tendency to aggregate, and the high frequency of post-transcriptional charge modification. The most widely used analytical system for histones; polyacrylamide gel electrophoresis at low pH resolves five major histone species and some of their modified forms. The resolution of the histones can be improved by the addition of nonionic detergents to the gels that results in a differential reduction in the electrophoretic mobility of different histones. This effect is because of the formation of mixed micelles between the detergent and the hydrophobic regions of protein molecules and is extremely sensitive to small differences in the hydrophobic properties of the proteins. For qualitative comparison of complex protein mixtures, the electrophoresis system can be combined with a simple and effective two-dimensional electrophoresis technique that uses the same buffer system in both directions and therefore avoids the precipitation problems and the possibility of differential loss of proteins during the transition from one buffer system to another.

The progeny of rabbit articular chondrocytes synthesize collagen types I and III and type I trimer, but not type II. Verifications by cyanogen bromide peptide analysis.

Abstract: The radioactive collagens synthesized by the fourth subculture progeny of rabbit articular chondrocytes were extracted and purified after limited pepsin digestion by neutral and acid salt precipitation. In order to identify the different types of collagen present, denatured collagen chains were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5% gels, electrophoretically eluted, and cleaved with cyanogen bromide, and the resultant peptides were fractionated by a new sodium dodecyl sulfate electrophoresis system (tris(hydroxymethyl)aminomethane-borate buffer, 15% gels). Comparison of these separate peptide profiles with those from alpha1(I) and alpha1(III) collagen chains permitted the unambiguous identification of these chains in the radioactive collagen synthesized by chondrocytes. Although cartilage slices predominantly synthesized alpha1(II) chains, only alpha1(I) chains were made by cells in fourth subculture. A large fraction of these alpha1(I) chains could not be accounted for by the presence of type I collagen. While in a native, triple-helical conformation, some of these extra alpha1(I) chains were completely separated from type I collagen by their solubility at pH 8.0 in 2.6 M NaCl and therefore identified as [alpha1(I)]3, type I trimer. In addition to type I collagen and type I trimer, these chondrocyte progeny also synthesized type III collagen and two new collagen chains, X and Y. Each collagen type was further characterized by carboxymethylcellulose chromatography and its distribution between the medium and the cell layer. These findings support the idea that cultured chondrocytes assume a collagen phenotype similar to that of their undifferentiated mesenchymal cell precursors.