Biotechniques

This assay is based on carboxyfluorescein labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases.

Experimental Preparation and Setup:

Working Dilution of FMK-peptide inhibitors:

1. Reconstitute lyophilized FMK-peptide in 50 µl of DMSO resulting in a 150X concentration.
2. Mix contents at room temperature until dissolved. Aliquots may be made and stored frozen at –20°C.
3. Prior to using, make a 30X Working Dilution. Dilute the 150X substrate 1:5 in PBS, pH 7.4 (1 part 150X FMK-peptide and 4 parts PBS). Mix well.
4. Protect from light at all times.

1X Working Dilution Wash Buffer:

1. Place 10X Wash Buffer in a 37°C water bath for 30 minutes to dissolve precipitated protein and buffer salts.
2. Mix thoroughly.
3. Dilute 10 ml of 10X Wash Buffer in 90 ml of dH2O and mix thoroughly.