Recombinant Human SMAC/Diablo Protein, CF

• Jurkat E6 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
• Formulation Buffer: 25 mM HEPES, 0.1 M KCl, pH 7.5
• Recombinant Human SMAC/Diablo (rhSMAC) (Catalog # 789-SM)
• Recombinant Human XIAP BIR3 Domain (rhXIAP) (Catalog # 895-XB)
• 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 with NaOH
• Substrate: Ac-Asp-Glu-Val-Asp-AFC (DEVD-AFC) (MP Biomedicals, 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 250 nM stock of rhXIAP (BIR3) (MW: 13.0 KDa) in Formulation Buffer. 
3. Prepare a curve of rhSMAC (MW: 21.6 KDa ) in Formulation Buffer. Make the following serial dilutions: 50,000, 15,000, 7500, 3000, 1500, 600 and 300 nM. Note: High point may not be achievable depending on lot received.
4. 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.
5. Prepare reaction mixtures in tubes by combining 5 μL of each rhSMAC curve dilution, 5 μL of rhXIAP (BIR3), 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.
   3. rhXIAP (BIR3) only Conrol: 5 μL of Extraction Buffer,  5 μL of rhXIAP (BIR3), 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture.
6. Incubate for 60 minutes at 30 °C.
7. After incubation, add 100 μL of Assay Buffer to each vial for a five-fold dilution. Mix briefly.
8. Dilute Substrate to 100 μM in Assay Buffer.
9. In a plate load 50 μL of diluted incubated reaction mixtures and start the reaction by adding 50 μL of 100 μM Substrate.
10. Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively, in kinetic mode for 5 minutes.
11. Derive the 50% inhibiting concentration (IC₅₀) of rhSMAC by plotting normalized activity vs. reaction concentration of rhSMAC with 4‑PL fitting.
12. Normalized activity may be determined using the following equation:

Per Reaction:

• rhSMAC curve:  10,000, 3000, 1500, 600, 300, 120 and 60 nM