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Characteristics
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Dissolve the caspase inhibitor in high purity DMSO (>99.9%) before use to make a stock solution of 20 mM.
Method for assay of Biotin-VAD-FMK Inhibitor:
1. Grow and treat 1 x 10^6 cells at the appropriate dose and time to obtain 50 - 60% apoptosis.
2. Collect cells by centrifugation, remove supernatant and re-suspend cell pellet in an initial volume of 1/1,000 th of the original media volume (10 µL for 10 mL media).
3. Add 20 µL of 2X Biotin-VAD-FMK in MGD buffer. (Final Biotin-VAD-FMK concentration = 10 µM.)
4. Freeze/thaw 3X to lyse cells.
5. Incubate at 37°C for 15 minutes. Spin two minutes at 14K to remove cell debris.
6. Transfer supernatant to new tube, add 13 µL of 4X SDS-PAGE buffer and run all on 10% SDS-PAGE gel and transfer to PVDF membrane.
7. Block one hour and incubate with streptavidin-HRP (1/1,000) for four hours. Develop by ECL.
MGD Buffer:
50 mM NaCl
2 mM MgCl2
5 mM EDTA
10 mM HEPES pH 7.0
1 mM DTT
Protease Inhibitor
IMPORTANT NOTE for in vitro use: Our peptide inhibitors are synthesized as methyl esters to enhance cell permeability. In intact cells, the methyl groups are removed by endogenous enzymes. For in vitro experiments with purified enzymes, however, the methyl groups must first be removed by treating the inhibitor with esterase. A procedure is available upon request.
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