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Dissolve Caspase-1,4,5 Inhibitor in high purity (99.9%) DMSO before use.
For use on intact cells: 1. Prepare desired concentrated stock solutions as follows: 1 mg Z-WEHD-FMK in 5μl DMSO = 20 mM in 114 μl DMSO = 10 mM in 228 μl DMSO = 5 mM, etc.
2. Add 2μl of 10 mM stock solution to 1 ml culture medium containing cells such that the final concentration of DMSO is 0.2%. Levels of DMSO above this may cause some cellular toxicity, thus masking the effect of the Caspase-1,4,5 protease inhibitor. Adding 2 μl of a 10 mM stock solution to 1 ml of culture medium gives a final final Z-WEHD-FMK concentration of 20 μM. Typical final concentrations of inhibitor are 5-20 μM.
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. 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.
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