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Continuous enzymatic assay for histone lysine methyltransferases

Biotechniques. 2007 Nov;43(5):602, 604, 606 passim. doi: 10.2144/000112623.

Abstract

We describe a continuous peptide methylation assay using the Neurospora crassa Dim-5 histone H3 lysine 9 (H3K9) methyltransferase as a model system. The assay uses streptavidin FlashPlates coated with target peptide. Since no washing and pipeting steps were required after the addition of the enzyme/S-adenosyl-L-methionine (AdoMet) mixture to the microplate, a continuous readout of the reaction progress was possible. We show that this assay is highly reproducible (with errors in the order of +/- 3%). The continuous assay is well suited for the simultaneous analysis of up to 384 samples, thus allowing for a rapid screening of methylation rates of different substrates under different conditions or in the presence of inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Histone-Lysine N-Methyltransferase / metabolism*
  • Kinetics
  • Methylation
  • Neurospora crassa / enzymology*
  • Peptides / metabolism
  • Reproducibility of Results
  • S-Adenosylmethionine / metabolism

Substances

  • Peptides
  • S-Adenosylmethionine
  • Histone-Lysine N-Methyltransferase