Recombinant Human Livin beta/ML-IAP Protein, CF

Preparation of Cell Extracts

• Jurkat A3 wild type cells (ATCC # CRL-2570)
• Phosphate Buffered Saline (PBS, pH 7.4)
• Extraction Buffer: 50 mM HEPES-KOH (pH 7.5), 10 mM KCl, 5 mM EGTA, 1 mM MgCl₂, 0.2% CHAPS, 0.2 mM DTT
• Protease inhibitors: Cytochalasin B (Sigma # C6762), Chymostatin (Sigma # C7268), Leupeptin (Sigma # L8511), Antipain (Sigma # A6191), Pepstatin (Sigma # P4265), PMSF (Sigma # P7626)

Activation of Caspase in Cell Extracts

• 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 KOH
• Recombinant Human Livin beta /ML-IAP(rhLivin beta ) (Catalog # 787-LV)
• Dilution Buffer: 25 mM HEPES (pH 7.5), 0.1 M NaCl, 1 mM DTT
• Assay Buffer: 25 mM HEPES (pH 7.5), 0.5 mM EGTA, 5 mM DTT, 10% Glycerol
• Substrate: Ac-Asp-Glu-Val-Asp-AFC (DEVD-AFC, MP Biomedicals, Catalog # AFC138) 500 μM stock in DMSO
• EIA/RIA 96-well plate (Costar, Catalog # 3369) or equivalent
• Fluorescence plate reader (Molecular Devices Model # SpectraMax Gemini EM) or equivalent

Preparation of Cell Extracts

1. Pellet cells from culture media by centrifugation at 1000 x g for 10 minutes at 4 °C.
2. Wash 2 times with PBS. Centrifudge as above and count cells before the final spin.
3. Add protease inhibitors to Extraction Buffer immediately prior to use. Final concentrations: 10 μg/mL Cytochalasin B, 2 ug/mL Chymostatin, 2 μg/mL Leupeptin, 2 μg/mL Antipain, 2 μg/mL Pepstatin, 100 μM PMSF and 1 mM DTT
4. Solubilize the cells in ice cold Extraction Buffer at a density of 2 x 10⁸ cells/mL.
5. Thoroughly resuspend the pellet by gently pipetting up and down. Incubate on ice for 10 minutes.
6. Pipette 200 μL aliquots into chilled microcentrifuge tubes.
7. Snap freeze in liquid nitrogen and store at ≤-70 °C. (Note: Freeze immediately at ≤-70 °C if liquid nitrogen is unavailable to snap freeze)
   

 Activation of Caspase in Cell Extracts

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

1. Thaw cell extracts and centrifuge at 14,000 x g for 5 minutes at 4 °C. Transfer supernatants to chilled tubes and use within 1 hour.
2. Dilute rhLivin beta (MW: 32 kDa) to various concentrations in Dilution Buffer. Make an initial dilution series of: 25,000, 12,500, 5000, 2500, 1250, 250, 125 and 25 nM. The final concentration range will be 10,000 to 10 nM in 25 μl total reaction volume.
3. Add 10 μL of cell extract to a tube containing 2.5 μL Cytochrome c, 2.5 μL of dATP and 10 μL Livin beta  dilution.
4. Total (no Livin beta ) and inactive (no Livin beta, Cytochrome-c, or dATP) controls should be run for each assay making up the volume difference with the appropriate buffer.
5. Incubate samples in a 30 °C water bath for 30 minutes.
6. To each well of a 96-well plate, add in the following order, 85 μL Assay Buffer and 5 μL of extracts activated in the presence or absence of added Livin beta.
7. Start the reaction by adding 10 μL of 500 μM DEVD-AFC (50 μM final concentration).
8. Read at excitation and emission wavelengths 400 and 505 nm, respectively, in kinetic mode for 5 minutes.

9. Derive the 50% inhibiting concentration (IC₅₀) of rhLivin  beta by plotting RFU/min vs. concentration with 4-PL fitting.