Continuous enzymatic assay for histone lysine methyltransferases.
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.
Rathert
Philipp
P
Jacobs University Bremen, Bremen, Germany.
Cheng
Xiaodong
X
Jeltsch
Albert
A
D001681:Biological Assay / Q000379:methods*; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D009492:Neurospora crassa / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D015203:Reproducibility of Results; D012436:S-Adenosylmethionine / Q000378:metabolism
D010455:Peptides; D012436:S-Adenosylmethionine; D011495:Histone-Lysine N-Methyltransferase