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Comment
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Peptide-fluoromethyl ketone inhibitor of caspases. The FMK (fluoromethyl ketone, CH2F) inhibitor has several advantages over other types of derivatives: Penetrates cell membranes, Not toxic to cells, Irreversible inhibition.
Dissolve the caspase inhibitor in high purity DMSO (>99.9%) before use to make a stock solution of 20 mM.
For use on intact cells:
1. Prepare desired concentrated stock solutions as follows:
1 mg Z-VAD-FMK
in 10µl DMSO = 20 mM
in 21µl DMSO = 10 mM
in 42µl DMSO = 5 mM, etc.
2. Add 2 µL of above stock solutions to 1 mL of culture medium containing cells to give a fµl DMSO concentration of 0.2%. Levels of DMSO above this may cause some cellular toxicity, thus masking the effect of the ICE-protease inhibitors. Adding 2 µL of a 10 mM stock solution to 1 mL of culture medium gives a final Z-VAD-FMK concentration of 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. A procedure is available upon request.
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