Preparation of chromosomal protein A24 (uH2A) by denaturing gel filtration and preparation of its free nonhistone component ubiquitin by ion-exchange chromatography.
Chromosomal protein A24 (uH2A) is unique in that it comprises the nucleosomal core histone H2A in isopeptide linkage with the highly conserved, globular, and stable nonhistone protein ubiquitin. Some 10% of the chromatin complement of H2A is modified in this way and studies to elucidate a role for this modification have concentrated on observations requiring no purification of A24 due to the difficulty in isolating the protein in large and pure quantities. We describe a method for isolating A24 by chromatography on Pharmacia G-100 gel filtration medium under urea denaturing conditions. A24 prepared by this method is structurally intact and is available in the quantities required for studies of the behavior and influence of the protein on histone-histone, histone-DNA, and enzymatic interactions. In conjunction with this method we describe a procedure for the isolation of large quantities of free ubiquitin of far greater purity than previously reported.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006609:High Mobility Group Proteins / Q000302:isolation & purification*; D006657:Histones / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D011489:Protein Denaturation; D013950:Thymus Gland / Q000032:analysis; D014452:Ubiquitins / Q000302:isolation & purification*
D000596:Amino Acids; D006609:High Mobility Group Proteins; D006657:Histones; D014452:Ubiquitins; C021645:chromatin conjugate protein A24