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J Biomol Screen. 2009 Oct;14(9):1129-33. doi: 10.1177/1087057109345528. Epub 2009 Sep 4.

A continuous protein methyltransferase (G9a) assay for enzyme activity measurement and inhibitor screening.

Author information

1
Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Bremen, Germany.

Abstract

The authors describe a continuous protein methylation assay using the G9a protein lysine methyltransferase and its substrate protein WIZ (widely interspaced zinc finger motifs). The assay is based on the coupling of the biotinylated substrate protein to streptavidin-coated FlashPlates and the transfer of radioactive methyl groups from the S-adenosyl-L-methionine to the substrate. The reaction progress is monitored continuously by proximity scintillation counting. The assay is very accurate, convenient, well suited for automation, and highly reproducible with standard errors in the range of 5%. Because of few pipetting steps and continuous data readout, it is ideal for high-throughput applications such as screening of inhibitors, testing many enzyme variants, or analyzing differences in methylation rates of different substrates under various conditions. By using this new assay, the IC(50) of AdoHcy and the G9a inhibitor BIX-01294 were determined for methylation of the G9a nonhistone substrate WIZ.

PMID:
19734436
DOI:
10.1177/1087057109345528
[Indexed for MEDLINE]

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