ASSAY PROTOCOL:

Materials: SIRT Assay Buffer (BPS Bioscience, # 50090) SIRT6 Substrate (BPS Bioscience, #50866) SIRT6 enzyme (BPS Bioscience, #50017) or other SIRT6-containing sample NUNC black 96-well plate, low protein binding (VWR #62408-936). SirtuinDeveloper (2X) (BPS Bioscience, #50089)

Step 1: Set up 50 µul of SIRT6 reactions containing SIRT assay buffer, 1 μg SIRT6 enzyme, and 5 µM SIRT6 substrate in the 96-well plate. Include a no-enzyme control reaction (“Blank”) to determine background fluorescence. Incubate at 37ºC for 30 minutes.

Step 2: Add 50 µl of SIRT Assay Developer (2x) (BPS #50089) and incubate the plate at room temperature for 15 minutes.

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. “Blank” value is subtracted from all other values.

Stable at least 6 months from date of receipt, when stored as directed at -20°C or -80°C

2. Rahnasto-Rilla, M., et al., 2016. Methods Mol Biol, 1436:259-269.