Preparative electrophoresis of histones in solubilizable polyacrylamide gels.
Histones from the mouse testis have been fractionated on 17% acrylamide gels containing 0.19% Bis-acrylylcystamine (BAC), 25 M urea and 0.4% Lubrol-WX at pH 2.7. Polyacrylamide gels which contain BAC, a cross-linking agent with disulfide bonds, can be solubilized in the presence of 2-mercaptoethanol, at pH 8.3. Polymerization is carried out at 40 degrees C and in the presence of 6.5--7.5% tetramethylethylenediamine (TEMED) at pH 8.3. Gels containing high acrylamide concentration (greater than 15%) are not soluble if polymerized at lower pH or TEMED concentrations. Following electrophoresis, the gels are stained with Coomassie brilliant blue R for the visualization of protein bands. The stained protein bands are excised and adjusted to pH 8.3 prior to their solubilization. The histones are recovered by ion-exchange chromatography and are free of soluble gel components and dye. Two variants of histone H2B (H2B . 1 and H2B . 2) have been isolated at approx. 95% purity using this single purification step.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003543:Cysteamine; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006657:Histones / Q000302:isolation & purification*; D008297:Male; D008623:Mercaptoethanol; D051379:Mice; D013737:Testis / Q000032:analysis*
D000596:Amino Acids; D006657:Histones; D003543:Cysteamine; D008623:Mercaptoethanol; D003545:Cysteine