Methods and tips for the purification of human histone methyltransferases.
Recently developed biochemical techniques have enabled researchers to study histone modifications more easily and accurately. One of these modifications, histone lysine methylation, has been shown to be highly stable and to represent an epigenetic alteration. Extensive biochemical analyses have led to discoveries about the nature and functions of this modification, thus accelerating our understanding of this crucial epigenetic event. Here we describe basic methods for purification and biochemical analysis of lysine-directed, histone methyltransferases from HeLa cell-derived extracts. In the section on substrate preparation, we describe a simple method for the preparation of recombinant substrates, although we recommend using native substrates for initial detection of the activities. The purification protocols for several histone methyltransferases have been streamlined so that those researchers with a basic understanding of biochemistry can perform them. We also describe many tips and provide suggestions to avoid common pitfalls in the biochemical analysis of histone methyltransferases.
Nishioka
Kenichi
K
Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA. knishiok@lab.nig.ac.jp
Reinberg
Danny
D
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D006367:HeLa Cells; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009707:Nucleosomes / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011496:Protein Methyltransferases; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014981:Xenopus
D009707:Nucleosomes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000076983:Histone Methyltransferases; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase