| Assay Method Information | |
| | HDAC-1, HDAC-2 and HDAC-6 Assays |
| Description: | The inhibitory effect of compounds on HDAC-1 and HDAC-6 function was determined in vitro using an optimized homogenous assay performed in 384-well plate format. In this assay, the recombinant, full-length HDAC-1, HDAC-2 or HDAC-6 protein (BPS Biosciences) was incubated with Ac-peptide-AMC with concentrations in the Km plot. Reactions were performed in Tris-based assay buffer and were followed for fluorogenic release of 7-amino-4-methylcoumarin from substrate upon deacetylase and trypsin enzymatic activity. Fluorescence measurements were obtained using a multilabel plate reader (Synergy MX with excitation at 355 nm and emission at 460 nm). Data were analyzed on a plate-by-plate basis for the linear range of fluorescence over time. Fit the data in GraphPad Prism V5.0 software to obtain IC50 values using equation (Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*Hill Slope), Y is % inhibition and X is compound concentration). |
| Affinity data for this assay | |
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