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Assay Method Information

Assay Name:  Enzymatic Assay
Description:  Biochemical activity of G9a was measured as described [Kubicek et al., Mol. Cell, 25:473-481]. Assays were performed in white, opaque 384-well plates coated with Neutravidin (Pierce Biotechnology). Brd 4770 was used as a positive control. Test compounds were diluted to 12 μg/ml in Baffer A (50 mM 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 Buffer B (50 mM 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 Buffer C (50 mM Tris pH 8.5) in a volume of 10 μL, and incubated at room temperature for 60 min. The plates were washed 3 times with 100 μL of Wash Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween 20, 0.2% BSA). Next, 50 μl of Fluoroimmunoassay (FI) Buffer (50 mM Tris HCl pH 7.8, 150 mM NaCl, 0.05% Tween 40, 25 μM DTPA, 0.2% BSA, 0.05% BGG) containing 5 ng α-2X-di-meth H3-K9 and 5 ng goat anti-rabbit Eu chelate (Perkin Elmer Life Sciences) was added to all wells of the plate, and the plate was incubated for an additional hour at room temperature. The plates were washed 3 times with 100 μL of Wash Buffer, and 50 μL of Enhancement Solution (Perkin Elmer Life Sciences) was added to each well. Time resolved fluorescence was measured after 45 min on a Viewlux Microplate Imager (Perkin Elmer Life Sciences) imaging for 15 s with a 354 μs window, 400 μs delay, excitation at 360 nm, and emission at 618 nm.
Affinity data for this assay
 

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Last update November 1, 2007
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