Assay Method Information

Assay Name:  Inhibitor Competition Assay
Description:  Following transfection of HEK293 cells with pBJ5-HDAC1 wild type or mutant plasmids, [Weerasinghe et al., J. Med. Chem. 51:5542-5551; Wambua et al., J. Med. Chem. 57:642-650] as described above, cells were grown for 48 h and then subsequently treated with SAHA (10 uM in growth media containing DMEM, 10% FBS, 1% antibiotic/antimycotic, and <2% DMSO) for another 24 h before harvesting. Cells (20 x 1^06) were lysed in lysis buffer (500 uL; 50 mM Tris-Cl at pH 8.0, 150 mM NaCl, 10% glycerol, and 0.5% triton-X100) containing 1x protease inhibitor cocktail (GenDEPOT) at 4 °C for 30 min with rotation. The supernatant was collected using centrifugation at 13.2 x 10^3 rpm for 10 min at 4 °C. Prior to immunoprecipitation, anti-FLAG agarose beads (20 uL bead slurry) were washed with cold TBS (tris buffered saline; 20 mM Tris-Cl at pH 8.0, 150 mM NaCl) two times with spinning at 5000 rcf for 1 min at 4 °C. Wild type or mutant HDAC proteins were immunoprecipitated using the prewashed anti-FLAG agarose beads by incubating at 4 °C overnight with rotation. For inhibitor competition experiments, SHI-1:2 (10 uM in lysis buffer) or tubastatin (10 uM in lysis buffer) was included during immunoprecipitation. After immunoprecipitation, beads were washed three times with lysis buffer (1 mL), and bound proteins were eluted by incubating for 30 min at 4 °C using 3x FLAG peptide (APEXBIO; 50 uL; 0.25 mg mL in TBS). The eluted proteins were mixed with 4x SDS loading dye (25 uL; 100 mM Tris-Cl at pH 6.8, 4% SDS, 20% glycerol, 0.008% bromophenol blue, and 10% v/v β-mercaptoethanol), separated by 10% SDS-PAGE, and visualized with Sypro Ruby total protein stain (Molecular Probes) according to the manufacturer's instructions.
Affinity data for this assay
 

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