Resolution of basic cellular proteins including histone variants by two-dimensional gel electrophoresis: Evaluation of lysine to arginine ratios and phosphorylation

A procedure is described which resolves histones and other very basic cellular polypeptides from solubilized whole cells by two-dimensional gel electrophoresis. The entry of histones into the gel was apparently quantitative when salt and protamine were added to solubilization buffers containing urea and detergents. Histones and basic polypeptides in the histone region of the gels were identified and characterized by comparison with purified histones and by determining lysine to arginine ratios of individual spots. Phosphorylated derivatives of the nucleosome histones were clearly resolved from stained spots in the charge dimension. Some phosphorylated H1's comgrated with the stained spots and some were retarded in the charge dimension. Acetylated nucleosome histones were nearly coincident with stained spots. The usefulness of this technique for evaluating changes in post-translational modification of histones was illustrated by showing that one H1-like protein increased in 32P content when C-6 cells were treated with a β-adrenergic agonist.