Isoelectric focusing of histones in extremely alkaline immobilized pH gradients: comparison with capillary electrophoresis

Various classes of calf thymus histones (fractions Il-AS, VI-S, VII-S and VIII-S) were separated to the steady state in an extremely alkaline immobilized pH gradient, covering non-linearly the pH 10-12 interval. Successful separations were obtained in 5%T, 4%C polyacrylamide matrices, reswollen in 8 M urea, 1.5% Tween 20, 1.5% Nonidet P-40 and 0.5% Ampholine pH 9-11. Additionally, in order to quench the very high conductivity of the gel region on the cathodic side, the reswelling solution contained a 0-10% (anode to cathode) sorbitol gradient. The best focusing was obtained by running the gel at 17°C, instead of the customary 10°C. All major histone components had pI values between pH 11 and 12 and only minor components (possibly acetylated and phosphorylated forms) focused below pH 11. By summing up all bands in Arg- and Lys-rich fractions, eight to ten major components and at least twelve minor zones are clearly resolved. In contrast, capillary zone electrophoresis (in a coated capillary. 7M urea. 50 mM Tris-acetate buffer, pH 8.0) can only resolve six major fractions and two minor, broad zones.