| Assay Method Information | |
| | Biochemical Assay |
| Description: | I. Compound handling: Testing compounds were dissolved in 100% DMSO to a specific concentration. The serial dilution was conducted by epMotion 5070 in DMSO. II. HDAC reaction buffer: 50 mM Tris-HCl, pH8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2, Added fresh: 1 mg/ml BSA, 1% DMSO. III. Substrate: Fluorogenic HDAC General Substrate for HDAC1, 2, 3, 6, 10, 11 ans Sirt1, 2 and 3: Arg-His-Lys-Lys(Ac); HDAC8 only substrate: Arg-His-Lys(Ac)-Lys(Ac); Class2A Substrate (HDAC4, 5, 7 and 9): Acetyl-Lys(trifluoroacetyl)-AMC; Sirt5 substrate: Ac-Lys(succinyl)-AMC. IV. General Reaction Procedure: (Standard IC50 determination) a. Delivered 2× enzyme in wells of reaction plate except No Enzyme (No En) control wells. Add buffer in No En wells. b. Delivered compounds in 100% DMSO into the enzyme mixture by Acoustic technology (Echo550; nanoliter range). Spin down and pre-incubation. c. Delivered 2× Substrate Mixture (Fluorogenic HDAC Substrate and co-factor (500 μM of Nicotinamide adenine dinucleotide (NAD+) in all Sirt assay) in all reaction wells to initiate the reaction. Spin and shake. d. Incubated for 1-2 hr. at 30° C. with seal. e. Added Developer with Trichostatin A (or TMP269 or NAD+) to stop the reaction and to generate fluorescent color. f. Fluorescence was read (excitatory, 360; emission, 460) using the EnVision Multilabel Plate Reader (Perkin Elmer) g. Endpoint reading was taken for analysis after the development reaches plateau. V. Data Analysis: The percentages of enzyme activity (relative to DMSO controls) and IC50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation. |
| Affinity data for this assay | |
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