Assay Method Information

Assay Name:  Kinetic Inhibition Assay
Description:  Purified CtBP2 in 50% glycerol was added to 150 μM NADH and variable amounts of MTOB and HIPP in buffer containing 25 mM HEPES, pH 7.1, 25 mM potassium chloride, and 1mM DTT. The final concentration of CtBP was 40 μg/mL (986 nM) per reaction. HIPP was dissolved in DMSO (1% total volume). Final MTOB concentrations were the following: 36, 24, 12, 8, 4, 2, 1, and 0 μM for 0 μM HIPP; 64, 48, 24, 12, 8, 4, 2, and 0uM for 500nM HIPP; 80, 64, 48, 24, 12, 8, 4, and 0 μM for 1 μM HIPP; 96, 80, 64, 48, 36, 24, 12, and 0 μM for 2 μM HIPP. Reaction components wereadded to 96-well UV-Star Microplates (Greiner Bio-One). Upon addition of CtBP,reactions were mixed vigorously and immediately read by a Synergy H1 microplatereader (BioTek). Absorbance was recorded at A=340nm every 7 seconds for 7 minutes at 25C to measure CtBP dehydrogenase function (NADH, but not NAD+ absorbs light at 340nm).
Affinity data for this assay
 

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