Materials
- 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
- 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.
- Dilute rhCaspase-7 to 0.12 ng/µL in Activation Buffer.
- 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
- Include a control, in duplicate, containing Assay Buffer and rhCaspase-7 only.
- Incubate at RT for 15 minutes.
- Dilute Substrate in Assay Buffer to 500 µM.
- Load 10 µL Substrate per well to start the reaction.
- Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively in kinetic mode for 5 minutes.
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Derive the 50% inhibiting concentration (IC50) for rhXIAP-BIR2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
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The specific activity for rhCaspase-7 at each point may be determined using the following formula (if needed):
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-trifluoromethylcoumarin (AFC) (Calbiochem, Catalog # 164580).
Per Well:
- 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
