Assay Method Information
Assay Name:  Scintillation Proximity Assay (SPA) Assay B (Human Truncated DNMT1(601-1600))
Description:  This assay used Scintillation Proximity technology in a signal increase format to evaluate the potency of compounds. Human truncated DNMT1(601-1600), single hemi-methylated CpG site oligonucleotide, and Tritiated SAM were utilized to monitor activity. Assay plate creation consisted of the following parameters: 10 mM Compounds (11-point, 3-fold serial dilution) were stamped at 100 nL per well (100× in 100% DMSO) into a Griener white LV 384 well plate (#784075). Assay buffer mix was made on the day of assay consisting of: base buffer: (500 mM Hepes, ph 8, 1M MgCl2 made in advance, stored at room temp as a stock), 10% NP40-Surfact AMPS, 10% Ultrapure BSA 50 mg/ml, and 2M DTT (DL-Dithiolthreitol). The 2× enzyme mix was then prepared consisting of: DNMT1 protein (truncate human DNMT1—601-1600, made in house at 16.876 uM stock concentration) added to the assay buffer mix. The 2× substrate mix was made last, and consists of: 1 mM 40-mer hemi-methylated DNA Oligonucleotide, 12.5 uM 3H-SAM (Adenosyl-L-Methionine-S-[methyl-3H] Specific Activity 55-85 Ci/mmol) and 32 mM solution of S-Adenosyl-L-Methionine (this was diluted to 1 mM in Nuclease Free-H2O before adding to substrate mix) added into the assay buffer mix (3H-SAM is added last). Five uL of the assay buffer mix was dispensed into column 18 ONLY using a Thermo Multidrop combi. Next, 5 uL of the 2× Enzyme mix was dispensed to columns 1-17, 19-24 using a Thermo Multidrop combi. Then 5 uL of the 2× Substrate mix was dispensed to the full plate using a Thermo Multidrop combi. Plates were stacked and incubated for 40 minutes with a cover plate over the top plate. The quench mix was made around the 25 minute mark of the incubation step, which consisted of: 32 mM solution of S-Adenosyl-L-Methionine & PerkinElmer PEI PS Imaging Beads (Cat. #RPNQ0098)(10 mg/ml) into Nuclease Free-H2O. The quench mix was vortexed prior to use to get the beads in solution. After the 40 minute incubation, 10 uL of the quench mix was dispensed to the full plate using a Thermo Multidrop combi. Plates were sealed with a clear seal and centrifuged at 1000 rpm/1 min and dark adapted for 30 minutes. Plates were read on a Viewlux (PerkinElmer, 613 nm emission filter, 300 sec dual-exposure, (10 min. total read time)).
Affinity data for this assay
 
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