SIRT Assay Developer is an essential reagent for signal generation in sirtuin assays performed with any of BPS Bioscience’s SIRT substrate or fluorimetric assay kits. An appropriate fluorimetric substrate, comprising an acetylated lysine side chain, is first incubated with a sample containing SIRT activity (nuclear or cellular extract, purified enzyme, bead-bound immunocomplex, etc.). Deacetylation of the substrate sensitizes it so that, in a second step, treatment with the SIRT assay developer produces a fluorophore. Nicotinamide is included as an ‘inhibitor stop’ for Sirtuins.

A 2x concentrate. Product is to be used (undiluted) at 1:1 ratio with SIRT reaction.

Aqueous buffer solution

In a 50 µL reaction, assay SIRT activity using SIRT (enzyme, lysate, etc.), substrate, SIRT assay buffer, and test inhibitor. Incubate microtiter plate at 37°C for 30 minutes. Add an equal volume (50 µL) of SIRT Assay Developer to each well. Incubate the plate at room temperature for 15 minutes. Read sample in a microtiter-plate reading fluorimeter capable of excitation at a wavelength in the range of 350-380 nm and detection of emitted light in the range of 440-460 nm. “Blank” value (no enzyme negative control) is subtracted from all other values.

Store, long term, only as the 2x concentrate, at -70°C. Concentrate is stable for at least 3 freeze-thaw cycles, but care should be taken that both freezing and thawing steps are done quickly (e.g. liquid N₂ or dry ice/EtOH for freezing, rapid finger rubbing for thawing). Frozen storage of smaller aliquots is recommended.

Study of sirtuin kinetics, regulation and inhibitor sensitivity. Ideal for drug discovery and HTS applications and cell-based SIRT assays.

1. K. Ito et al. Proc. Natl. Acad. Sci. USA 2002 99 8921.
2. B. Heltweg and M. Jung Anal. Biochem. 2002 302 175.
3. S. Milutinovic et al. J. Biol. Chem. 2002 277 20974

Avoid freeze/thaw cycles. Use product undiluted.

Deacetylase

Kit Reagent