Hat activity assay kit

20 μg of nuclear extracts from sham and ssUV radiated cells were used to analyze the enzyme activities of histone acetylatransferase (HAT) and deacetylase (HDAC). HAT activity was analyzed by HAT activity assay kit (Active Motif, Carlsbad, CA) using either H3 or H4 peptides as the substrates. The nuclear extracts were incubated with either histone H3 or H4 peptides in a mixture containing acetyl-CoA for 1 hour at room temperature. After the reaction was terminated with stop solution, the samples were incubated with developing solution for additional 15 minutes. The fluorescence in each sample was assessed with excitation wavelength at 360 nm and emission wavelength at 460 nm using a Spectra Max Gemini (Molecular Devices). HDAC activity was measured using the fluorescent HDAC assay kit (Active motif, Cartsbad, CA). For specify the different classes of HDAC, Trichostatin A (TSA) (Sigma, St. Louis, MO) were used to inhibit class I/II HDACs. Briefly, the nuclear extracts were incubated with HDAC substrate in 96-well plate at 37°C for 60 minutes, and followed by incubation with HDAC developer solution for additional 15 minutes. The fluorescence was assessed at an excitation wavelength of 365 and emission wavelength of 460 nm.

HAT activity was performed by using a non-radioactive HAT activity assay kit (Active Motif, United States), as per manufacturer’s instructions. Briefly, the nuclear protein (3 μg) was incubated in HAT assay buffer containing mixture of acetyl-CoA and histone H3 peptide for 30 min at RT. The reaction was terminated by stop solution. The sample was incubated with developing solution for 15 min at RT. Fluorescence (excitation, 380 nm; emission, 460 nm) was measured using a microplate reader (Tecan Group Ltd, Switzerland). HAT activity was expressed as arbitrary fluorescence units (AFU).