Recombinant Human cIAP-1 (HIAP-2) Protein, CF

• Jurkat A3 wild type cell extracts (see supplementary methods for preparation)
• Extraction Buffer: 50 mM HEPES, 10 mM KCl, 5 mM EGTA, 1 mM MgCl₂, 0.2% CHAPS, 0.2 mM DTT, pH 7.5
• Assay Buffer: 10 mM HEPES, 0.5 mM EGTA, 5 mM DTT, 10% Glycerol, pH 7.5
• Recombinant Human cIAP‑1/HIAP‑2 (rhcIAP-1) (Catalog # 818-IA)
• Cytochrome C, Bovine heart (Sigma, Catalog # C3131), 2 mg/mL stock in deionized water
• dATP (Sigma, Catalog # D6500), 10 mM stock adjusted to pH 7.5
• Cell Extracts from Jurkat A3 wild type cells (see above protocol)
• Substrate: Ac-Asp-Glu-Val-Asp-AFC (DEVD-AFC) (MP Biomendicals, Catalog # AFC138), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Note: All reagents and assay components should be kept on ice until use.

1. Thaw cell extracts and centrifuge in a microcentrifuge at 14,000 rpms for 5 minutes at 4 °C. Transfer supernatants to chilled tubes and use within 1 hour.
2. Prepare a curve of rhcIAP-1 (MW: 72,000 Da) in Extraction Buffer. Make the following serial dilutions: 5000, 2000, 1000, 500, 250, 125, 62.5, and 12.5 nM. Note: High point may not be achievable depending on lot received.
3. Prepare the activator mixture by combining equal volumes of 2 mg/mL Cytochrome C and 10 mM dATP for working concentrations of 1 mg/mL and 5 mM, respectively.
4. Prepare reaction mixtures in tubes by combining 10 μL of each rhcIAP-1 curve dilution, 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture. Also, prepare the following controls:
1. Total Control: 10 μL of Extraction Buffer, 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture.
2. Inactive Control: 15 μL of Extraction Buffer and 10 μL of cell extract supernatant.  The total reaction volume is 25 μL.
5. Incubate for 60 minutes at 30 °C.
6. After incubation, add 100 μL of Assay Buffer to each vial for a 1/5 dilution. Mix briefly.
7. Dilute Substrate to 100 μM in Assay Buffer.
8. In a plate load 50 μL of diluted incubated reaction mixtures, and start the reaction by adding 50 μL of 100 μM Substrate.
9. Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively, in kinetic mode for 5 minutes.
10. Derive the 50% inhibiting concentration (IC₅₀) of rhcIAP-1 by plotting normalized activity vs. reaction concentration of rhcIAP-1 (step 4) with Semi-Log Fitting [y = A + B * Log(x)].  Solve for x when y = 50.
11. Normalized activity may be determined using the following equation:

% Normalized Activity =	Sample (RFU/min) - Inactive Control** (RFU/min)	x 100%
	Total Control (RFU/min)

Per Reaction:

• rhcIAP-1 curve:  2000, 800, 400, 200, 100, 50, 25, and 5 nM
• Substrate: 50 μM