A fluorogenic, acetylated peptide substrate for SIRTs. It is a suitable substrate for SIRT1, SIRT2 and SIRT3 (tested so far). The substrate is based on residues 379-382 (Arg-His-Lys-Lys) of p53, a site of regulatory acetylation by the p300 and CBP acetyltransferases on lysines 381 and 382. This substrate fluoresces at excitation 350-380 nm, emission at 440-460 nm. The substrate should be used in conjunction with SIRT Developer.

5 mM DMSO solution (100 µl)

DMSO solution

Materials :
SIRT Assay buffer (BPS catalog #50090)
SIRT Assay developer (BPS catalog #50089)
SIRT Substrate (BPS catalog #50080)
SIRT1 (1 µg/µl) (BPS catalog #50012) or other SIRT enzyme

Step 1 :
Add the following reagents to a low binding NUNC black plate (VWR catalog #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 by adding 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.

Stable at least 12 months from date of receipt, when stored as directed

Study enzyme kinetics, and screen small molecule sirtuin inhibitors for drug discovery and HTS applications.

1. A. Ito et al. (2001) EMBO J. 20 1331.
2. A. Ito et al. (2002) EMBO J. 21 6236.
3. N.A. Barlev et al. (2001) Mol. Cell 8 1243.

Application Reference:
SIRT2 Interacts with β-Catenin to Inhibit Wnt Signaling Output in Response to Radiation-Induced Stress (2014)

Protect from direct exposure to light. Avoid freeze/thaw cycles.

Deacetylase

Substrate