EIA/RIA 96-well plate (Costar, Catalog # 3369) or equivalent
Fluorescence plate reader (Molecular Devices Model # SpectraMax Gemini EM) or equivalent
Note: All reagents and assay components should be kept on ice until use.
Thaw cell extracts and centrifuge at 14,000 x g for 5 minutes at 4 °C. Transfer supernatants to chilled tubes and use within 1 hour.
Dilute rhXIAP-BIR2 (MW: 14.8 kDa) to various concentrations in Dilution Buffer. Make an initial dilution series of: 25,000, 12,000, 5000, 2500, 500, 250, 50, 25, and 5 nM. The final concentration range will be 10,000 to 2 nM in a 25 µL total reaction volume.
Add 10 µL of cell extract to a tube containing 2.5 µL Cytochrome c, 2.5 µL dATP and 10 µL rhXIAP-BIR2.
Total (no rhXIAP-BIR2) and inactive (no rhXIAP-BIR2, Cytochrome c, or dATP) controls should be run for each assay making up the volume difference with the appropriate buffer.
Incubate samples in a 37 °C water bath for 60 minutes.
To each well of a 96-well plate, add in the following order, 80 µL Assay Buffer and 10 µL of extracts activated in the presence or absence of added rhXIAP-BIR2.
Start the reaction by adding 10 µL of 500 µM DEVD-AFC (50 µM final concentration).
Read at excitation and emission wavelengths 400 and 505 nm, respectively, in kinetic mode for 5 minutes.
Derive the 50% inhibiting concentration (IC50) of rhXIAP-BIR2 by plotting RFU/min vs. concentration with 4-PL fitting.
The IC50 for inhibition of DEVD-AFC cleavage in activated cell extracts is typically 50-300 nM.