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 (Full length; MW: 58 kDa) to various concentrations in Dilution Buffer. Make an initial dilution series of: 2,500, 1,000, 500, 250, 100, 50, 25 and 5 nM. The final concentration range will be 1,000 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 XIAP.
Total (no rhXIAP) and inactive (no rhXIAP, 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.
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 by plotting RFU/min vs. concentration with 4-PL fitting.
The IC50 for inhibition of DEVD-AFC cleavage in activated cell extracts is typically 40-100 nM.