Protein a b plus agarose beads

Total HDAC activity was measured using an Epigenase HDAC Activity/Inhibition Direct Assay Kit (Colorimetric) (EpiGentek, NY, USA). For pan HDAC activities, the protein extracts prepared as described above were added into a 96-well ELISA plate coated with acetylated substrate peptide at 10 μg protein in 49 μl of HDAC assay buffer and 1 μl of substrate per well in duplicates. For detection of specific HDAC activities, HADC1, HDAC2 or HDAC3 were immunoprecipitated from 100 μg of protein extracts using 4 μl of specific Abs as described by us (Wang et al., 2012 (link)) using protein A/B plus agarose beads (Santa Cruz Biotech) after preclearing the proteins extracts with protein A/B plus agarose beads. The immunoprecipitated proteins were assayed for HDAC activity using equal volumes in HDAC assay buffer in duplicates. The plate with protein extracts or specific HDACs was incubated at 37°C for 90 minutes. The plate was washed and incubated with capture and detection Ab and developed following the protocol provided by the manufacturer. The activity of HDAC (OD/min/mg protein) was calculated according to manufacturer’s specifications based on duplicate measurements of the optical densities at 450 nm.