A fluorogenic, acetylated peptide substrate for HDACs and SIRTs. Based on residues 379-382 of p53 (Arg-His-Lys-Lys(Ac), a site of regulatory acetylation by the p300 and CBP acetyltransferases (lysines 381, 382)1-3, it is a suitable substrate for HDAC2, HDAC3, HDAC6; SIRT1, SIRT2 and SIRT3 tested so far. This substrate should be used in conjunction with HDAC or SIRT Developer, respectively.
Step 1: Adding all reaction mixture to a low binding NUNC black plate (VWR catalog number 62408-936) 1 μl of HDAC2 (0.1 μg/μl) 39 μl of HDAC assay buffer 5 μl of 1 mg/ml BSA 5 μl of 200 μM substrate Incubate at 37 ºC for 30 min.
Step 2: Stop the reaction Add 50 μl of HDAC assay developer (2x) (BPS catalog # 50030) and incubate the plate at room temperature for 15 min.
Step 3: Read sample in a microtiter-plate reading fluorimeter capable of excitation at a wavelength in the range 350-380 nm and detection of emitted light in the range 440-460 nm.
Step 1: Adding all reaction mixture to a low binding NUNC black plate (VWR catalog number 62408-936) SIRT Assay Buffer, 35 µl 1 mg/ml BSA, 5 µl 200 µM SIRT substrate, 5 µl SIRT1, 5 µl Incubate at 37°C for 30 min.
Step 2: Stop the reaction Add 50 µl of SIRT assay developer (undiluted) and incubate the plate at room temperature for 15 min.
Step 3: Read samples in a microtiter-plate reading fluorimeter capable of excitation at 350-380 nm and emission at 440-460 nm. “Blank” value (no enzyme negative control) is subtracted from all other values.
Applications
Study enzyme kinetics, and screen small molecule HDAC inhibitors for drug discovery and HTS applications.
Shipping Temperature
-20°C or-80°C
Warnings
Protect from direct exposure to light. Avoid freeze/thaw cycles.