A new procedure for the purification of rat testis-specific histone TH2B involving affinity-related chromatography

Abstract

A new procedure for the isolation of rat testis-specific histone TH2B has been devised. First, rat testis chromatin fragments were applied to a hydroxylapatite column in 0.5 m NaCl, 0.1 m potassium phosphate buffer, pH 6.7, and histones were selectively stripped off the bound DNA in groups (H1/TH1, H2A/H2B/TH2B, and H3/H4). The fraction containing H2A, H2B, and TH2B, but lacking H3, was reduced, desalted, and applied to a p-chloromercuribenzoyl-aminoethyl-Sepharose 4B-CL column in 8 m urea, 0.1 m Tris-HCl, pH 8.1. After washing with the same buffer to remove H2A and H2B, covalently bound TH2B was eluted out with 10 mm dithiothreitol in the same buffer. No contaminants were detectable in the purified TH2B either by polyacrylamide gel electrophoresis in 0.4% Triton X-100, 2.5 m urea, 0.9 n acetic acid, or by N-terminal analysis with dansyl chloride. © 1981.