| Assay Method Information | |
| | Enzymatic Assay |
| Description: | The following describes an assay protocol for measuring the deacetylation of a peptide substrate by the enzymes HDAC2 or HDAC1. Enzyme, substrate, and cofactors are combined in a well of a microtiter plate and incubated for 3 hours at 25° C. At the end of the incubation, the reaction is quenched by the addition of an SDS-containing buffer. Substrate and product are separated and quantified electrophoretically using the microfluidic-based LabChip 3000 Drug Discovery System from Caliper Life Sciences. The peptide substrate used in this assay is FAM-TSRHK(AC)KL-CONH2 (FAM is carboxyfluorescein). Peptide should be >98% purity by Capillary Electrophoresis. 1. To a well of a 384-well plate, add 5 μL of 2× enzyme buffer. Using Labcyte Echo 550, add 100 nl compound. Enzyme and compound may be pre-incubated at this time if desired. 2. Add 5 μL of 2× substrate buffer. 3. Incubate plate at 25° C. for 17 hours. 4. Terminate reaction by adding 40 μL of 1.55× stop buffer. 5. Create job on a Caliper LabChip 3000 Drug Discovery System. 6. Load the plate and start electrophoresis using blue laser (480 nm) for excitation and green CCD (520 nm) for detection (CCD2). Reaction time=17 hours; Reaction temperature=25° C.Final Assay Reaction Mixture100 mM HEPES, pH 7.5 0.1% BSA 0.01% Triton X-100 25 mM KCl1% DMSO (from compound) 1 μM FAM-TSRHK(AC)KL-CONH2 5 nM HDAC Enzyme (specific activity may vary from lot-to-lot, and enzyme concentration may need to be adjusted to yield 10-20% conversion of substrate to product). |
| Affinity data for this assay | |
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