Recombinant Human XIAP (Full Length) 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% (w/v) CHAPS, 10% Sucrose, pH 7.5
• Assay Buffer: 25 mM HEPES, 100 mM NaCl, pH 8.0
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Recombinant Human Caspase‑7 (rhCaspase-7) (Catalog # 823-C7)
• Recombinant Human XIAP Full Length (rhXIAP) (Catalog # 822-XF)
• Substrate:  Ac-Asp-Glu-Val-Asp-AFC (MP Biomedicals, Catalog # AFC138), 10 mM stock in DMSO

1. Dilute rhCaspase-7 to 0.2 ng/µL in Activation Buffer.
2. Dilute rhXIAP (MW: 58,000 Da) to various concentrations in Assay Buffer. Make serial dilutions of: 12000, 5000, 1500, 500, 150, 50, 15, 5 and 1 nM.
3. Load Plate with the following:
      10 µL of the rhXIAP dilution curve
      10 µL of 0.2 ng/µL rhCaspase-7
      70 µL Assay Buffer
4. Include a control, in duplicate containing Assay Buffer and rhCaspase-7 only.
5. Incubate plate at RT for 15 minutes.
6. Dilute Substrate to 500 µM in DMSO.
7. Load 10 µL Substrate per well to start 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) of rhXIAP by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
10. The specific activity of rhCaspase-7 at each point may be determined using the following equation (if needed):

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

• rhCaspase-7: 0.002 µg
• rhXIAP: 1200, 500, 150, 50, 15, 5, 1.5, 0.5, and 0.1 nM
• Substrate: 50 µM