Hdac assay developer

The HDAC2 assay kit^{24 (link)} was purchased from BPS Bioscience (San Diego, CA). The assays were carried out in black background. Each well contained a volume of 50 μL including 30 μL buffer (BPS Bioscience, Catalog No. 50031), 5 μL BSA (1 mg/mL, Sigma-Aldrich), 5 μL inhibitor (1000, 631, 398, 251, 158, and 100 μM), 5 μL HDAC2 (1.8 ng/μL, BPS Bioscience, Catalog No. 50002), and 5 μL HDAC substrate (200 μM, BPS Bioscience, Catalog No. 50037). Prior to adding substrate, the plate was pre-incubated at 37 °C for 30 minutes. Upon the addition of substrate the plate was incubated at the same temperature for another 30 minutes. The HDAC assay developer (50 μL, BPS Bioscience, Catalog No. 50030) was then added to each well and the plate was incubated for another 15 minutes. The fluorescence was measured at excitation and emission wavelengths of 360 nm and 460 nm, respectively. We had the negative control containing protein without inhibitors and the positive control with TSA inhibitor. In addition, the readout of blank well containing no inhibitor and protein was subtracted from each well in measurement. All the assays were performed in triplicate.

The HDAC assay kit was purchased from BPS Bioscience (San Diego, CA). The assays were carried out in black NUNC 96-well plates. Each well contained a volume of 50 μl including buffer (BPS Bioscience, catalogue no. 50031), the mixture of proteins (HDAC-1 (0.033 mg/ml, BPS Bioscience, catalogue no. 50051) or HDAC-6 (0.025 mg/ml, BPS Bioscience, catalogue no. 50006) and competing proteins), inhibitor (at the IC50), BSA (1 mg/mL, Sigma-Aldrich), and HDAC substrate 3 (20 μM, BPS Bioscience, catalogue no. 50037). Upon addition of substrate, the plate was incubated at 37 °C for 30 min. HDAC assay developer (50 μL, BPS Bioscience, catalogue no. 50030) was then added to each well and the plate incubated for 15 min at room temperature. The fluorescence was recorded at excitation and emission wavelengths of 360 and 460 nm, respectively. Blank wells containing no inhibitor or protein were subtracted from all wells. The control wells, containing no inhibitor, were arbitrarily set as 100% activity. The assays were performed in triplicate, and each assay contained each inhibitor/competing protein combination was conducted three times. The data were normalized to values measured for uninhibited enzyme. Assays were reported as mean ± standard deviation.