HDAC Assay Developer (2X)

SIRT Assay Developer is an essential reagent for signal generation in sirtuin assays performed with BPS Bioscience?s SIRT 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. Applications: Study of sirtuin kinetics, regulation and inhibitor sensitivity. Ideal for drug discovery and HTS Applications and cell-based SIRT assays. Supplied as: A 2x concentrate. Usage: 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. Storage: -70°C. 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.