Focusing In Slab Gels Research Articles

Quantitation of proteins separated in N,N′-1,2-dihydroxyethylenebisacrylamide-crosslinkedpolyacrylamide gels

A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N,N′-1,2-dihy-droxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 μg and not disturbed by dissolved gel. Separations in N,N′-1,2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels.

Hemoglobin biosynthesis in individual bursts from human adult peripheral and umbilical cord blood: analysis of the relative rates of synthesis of G gamma and A gamma globin chains.

We cultured human adult peripheral and umbilical cord blood mononuclear cells in methylcellulose and measured the synthetic rates of globin chains in individual erythropoietic bursts. Globin chains were labeled with 14C-amino acids in culture, separated by isoelectric focusing in slab gels containing 8 M urea and 3% Nonidet P-40, and quantitated by fluorographic methods. All bursts exhibited G gamma, A gamma, and beta chains in varying ratios. Cumulative frequency distributions of individual bursts differing in the ratios of G gamma/(G gamma + A gamma) and gamma/(gamma + beta) approached normal distributions. These results indicated that perinatal hemoglobin switching and the hemoglobin switching of cultured adult erythropoietic precursors do not involve population changes in the precursors. Further, a positive correlation between gamma/(gamma + beta) and G gamma/(G gamma + A gamma) ratios of individual bursts existed in all of the samples. Switching between gamma and beta chains and switching of G gamma: A gamma ratios may share common regulatory mechanisms.

A new electrophoretic-autoradiographic method for the visual detection of phosphotransferases

In this paper we present the details of a slab acrylamide, lanthanum precipitation, autoradiographic technique and show it to be useful for the visualization of at least four different enzymes. We believe that with appropriate separation conditions and reaction mixtures this technique could be extended to a larger number of enzymes, theoretically all those whose isotopically labeled product could be specifically precipitated within the matrix of a polyacrylamide gel. It will be interesting to use this technique with other gel buffer systems, particularly those with a lower pH that have recently been reported (21). In addition, it might be useful to combine it with isoelectric focusing in slab gels (10). The technique would appear to be particularly useful for phosphotransferases and, to date, it has been applied to thymidine kinase and adenosine kinase with encouraging results. Work with other enzymes, and adapting the technique to starch gels, is also in progress.