Histone acetyltransferase activity assay kit

HAT activity was measured using a Histone Acetyltransferase Activity Assay Kit (Colorimetric) (ab65352, Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, approximately 50 mg of the oocyte samples at 0, 8, and 28 HPO and HPS were homogenised in 0.1% Triton X-100 on ice and then centrifuged at 10,000× g for 5 min at 4 °C. The supernatant was used for histone acetyltransferase activity assays. The total protein concentration was estimated using the Bio-Rad protein assay kit (500-0001, Bio-Rad Laboratories, Hercules, CA, USA). Fifty micrograms of protein extract were incubated with HAT substrate I, HAT substrate II, and NADH-generating enzyme in HAT assay buffer for up to 4 h at 37 °C. An ELISA plate reader (Synergy 2, BioTek Instruments, Winooski, VT, USA) was used to measure the absorbance at 440 nm. The active nuclear extract was used as a positive control and a standard. All measurements were performed in duplicate.

Relative HAT activity was quantified in PN1 DG with the Histone Acetyltransferase Activity Assay Kit (Abcam) according to the manufacturer’s instructions. This kit measures total HAT activity using strip wells coated with peptide substrate and a solution with the cofactor acetyl-CoA. Acetylation of the peptide substrate by functional HATs initiates release of the free form of CoA that acts as an essential coenzyme for production of NADH. NADH is then detected spectrophotometrically following reaction with a soluble tetrazolium dye. Samples of whole-cell tissue extracts (40 μl of 1 μg/μl) were incubated with assay mix at 37 °C for 1 h. The plate was assessed in a microplate reader at 440 nm. Data was analyzed as the relative O.D. value per μg.