| Assay Method Information | |
| | HDAC1 & HDAC3 Inhibition Assay |
| Description: | IC50 determination for HDAC1 and HDAC3—Prior to adding reagents, 50 nL/well of compound were added to Proxiplates using an Echo dispenser instrument (Beckman Coulter, CA, US). After addition of compound, 2.5 μL/well of 2× enzyme solution was added and the enzyme was pre-incubated with compound for 3 hours at room temp. (˜22° C.). After the pre-incubation period, the enzyme reaction was initiated by adding 2.5 μL/well of 2× Fluor de Lys® substrate solution. The assay reaction was stopped after one hour by adding 5 μl/well of a 0.66× Developer solution containing TSA (3.2 μM). The final product of the enzyme reaction was read using a Spectramax plate reader instrument (Molecular Devices, CA, US) using a fluorescent readout (Ex 360 nm/Em 460 nm). The final enzyme concentrations in the assay buffer for HDAC1 and 3 were 6 and 1.2 nM, respectively. The final Fluor de Lys® substrate concentration in the assay buffer was 16 μM for all HDAC enzymes. IC50 values were determined using the following equation:EfreeEo=100(1-11+(1IC50)n)(1)Where Efree and Eo are the free and total amount of HDAC enzyme in the reaction mixture, n is the hill coefficient, I is the free inhibitor concentration, and IC50 is the measure of the potency equivalent to the inhibitor concentration that leads to a 50% occupancy of the total enzyme. Each data was generated in duplicate. |
| Affinity data for this assay | |
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