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Dissolve Biotin-FA-FMK in high purity (>99.9%) DMSO before use.
For use on intact cells: 1. Prepare desired concentrated stock solution in DMSO as follows: Dissolve 1 mg Biotin-FA-FMK in 68 μl DMSO = 25 mM in 85 μl DMSO = 20 mM in 169 μl DMSO = 10 mM, etc.
2. Add 2 μL of the stock solution to 1 mL culture medium containing cells such that the final DMSO concentration is 0.2%. Levels of DMSO above this may cause some cellular toxicity, thus masking the true effects of the inhibitor. The recommended final concentration of Biotin-FA-FMK for use as a caspase inhibitor negative control is 50 μM (2 μL of 25 mM stock solution in 1 mL cell culture).
3. Detect bound Biotin-FA-FMK by any of several avidin/streptavidin methods.
For extended use in vivo or in vitro: For experiments extending 12 to 48 hours, fresh inhibitor may have to be added (injected) due to inactivation of the inhibitor by endogenous cysteine proteases.
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