Fig. 2. The p33/30 purified by heparin-affinity chromatography from myotubes is histone H1. (A) Triton X-100/KCl fractions (T) were subjected to heparin-affinity chromatography, and the resulting unbound (U) and NaCl-eluted fractions (E1-11) were analyzed by SDS-PAGE and stained with Coomassie blue. The molecular weight markers and the NaCl gradient are shown. Arrows on the right indicate the migration of p33/30. (B) A 15 amino acid sequence was obtained from a HPLC-purified p33-derived peptide. High homology with sequences of various subclasses of histone H1 from mouse, human, rat and bovine are shown for the peptide. Sequences are given in the one-letter code for amino acids. Underlined amino acids correspond to identities with the p33 sequence (in bold). (C) TX-100/KCl, PBS/heparin extracts, heparin-affinity purified p33/30 and an acid-soluble nuclear fraction obtained from myotubes were separated by 12.5% (20% in the case of nuclear extract) SDS-PAGE and stained with Coomassie blue (left panel). Similar SDS gels were transferred onto nitrocellulose membranes, stained with a specific monoclonal anti-histone H1 antibody and detected by ECL (right panel). (D) C₂C₁₂ myoblasts were extracted in the presence of 2 mg/ml heparin (Hep), chondroitin sulfate (CS), dermatan sulfate (DS) or N-desulfated heparin (N-deS Hep). Equivalent amounts of total proteins were analyzed by western blot with a specific monoclonal antihistone H1 antibody, and detected by ECL. Molecular weight markers are shown on the left and the migration of p33/30 is indicated (arrows) on the right of each panel.