HMTase Assays

Dissociation Enhanced Lanthanide Fluoro-Immuno Assay (DELFIA) was performed in white, opaque 384-well plates coated with Neutravidin. Test compounds were diluted to 12 μg/mL in 50mM Tris-HCl (pH 8.5) containing 4% DMSO and 10 μL was dispensed into the wells. Blank and control wells received only compound buffer. GST-G9a at 10 μg/mL and SAM at 40 μM were diluted in 50mM Tris HCl (pH 8.5)/10 mM DTT and added in a volume of 20 μL. Blank wells received Tris/DTT buffer only. The reactions were initiated by the addition of 800 nM H3(1-20)-cysbiotin substrate in 50 mM Tris (pH 8.5) in a volume of 10 μL, and incubated at room temperature for 60 mins. The plates were washed 3 times with 100 μL of Wash Buffer. Next, 50 μL of Fluoroimmunoassay (FI) Buffer containing 5 ng α-2X-di-meth H3-K9 and 5 ng goat anti-rabbit Eu chelate was added to all wells of the plate, and the plate was incubated for an additional 1 hr at room temperature. The plates were washed 3 times with 100 μL of Wash Buffer, and 50 μL of Enhancement Solution was added to each well. Time resolved fluorescence was measured after 45 mins on a Viewlux Microplate Imager imaging for 15 seconds.

Cell lines

Wild-type and G9a deficient mouse embryonic stem (ES) cells, mouse embryonic fibroblasts and human HeLa cells

Preparation method

The solubility of this compound in DMSO is > 10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 μM; over 2 days

Applications

BIX-01294 (4.1 μM) reduced the H3K9me2 Levels in Bulk Histone Preparations from wild-type mouse ES cells, mouse embryonic fibroblasts and human HeLa cells, but not in G9a deficient mouse ES cells.

References:

[1]. Kubicek S, O'Sullivan RJ, August EM, Hickey ER, Zhang Q, Teodoro ML, Rea S, Mechtler K, Kowalski JA, Homon CA, Kelly TA, Jenuwein T. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol Cell. 2007 Feb 9;25(3):473-81.