Recombinant Human XIAP (BIR2) Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Activation Buffer: 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS, 10% Sucrose, pH 7.5
• Assay Buffer: 25 mM HEPES, 0.1 M NaCl, 1 mM DTT, pH 8.0
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Recombinant Human Caspase‑7 (rhCaspase‑7) (Catalog # 823-C7)
• Recombinant Human XIAP BIR2 Domain (rhXIAP-BIR2) (Catalog # 786-XB)
• Substrate: Ac-Asp-Glu-Val-Asp-AFC (MP Biomedicals, Catalog # AFC138), 10 mM stock in DMSO

1. Dilute rhXIAP-BIR2 (MW: 14,801Da) to various concentrations in Assay Buffer. Make serial dilutions of: 8000, 2000, 1000, 300, 100, 50, 30, 10, 3, and 1 nM.
2. Dilute rhCaspase-7 to 0.12 ng/µL in Activation Buffer.
3. Load Plate in the following order:
   70 µL Assay Buffer
   10 µL of the rhXIAP-BIR2 dilutions
   10 µL of 0.12 ng/µL rhCaspase-7
4. Include a control, in duplicate, containing Assay Buffer and rhCaspase-7 only.
5. Incubate at RT for 15 minutes.
6. Dilute Substrate in Assay Buffer to 500 µM.
7. Load 10 µL Substrate per well to start the reaction.
8. Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively in kinetic mode for 5 minutes.

9. Derive the 50% inhibiting concentration (IC₅₀) for rhXIAP-BIR2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.

10. The specific activity for rhCaspase-7 at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-trifluoromethylcoumarin (AFC) (Calbiochem, Catalog # 164580).

• rhCaspase-7: 0.0012 μg
• rhXIAP-BIR2 curve: 800, 200, 100, 30, 10, 5, 3, 1, 0.3 and 0.1 nM
• Substrate: 50 µM