[박상규] caspase activity.

[Anonymous]

cells were collected and lysed in an ice-cold lysis buffer containing 20mM EPES at pH 7.5 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.5% NP-40 containing 0.1mM PMSF. The cell lysates were centrifuged at 15,000 Xg for 5 min at 4 degree and the supernatant fractions were used to quantitate caspase activity. Aliquots of 40 ug of cell lysate protein were incubated for 2h at 30 degree in assay buffer containing 20 mM HEPES at pH 7.5, 2 mM DTT, 10% glycerol and caspase substrates 100uM (1 : Ac-YVAD-pNA, 3 : Ac-DEVD-pNA, 8 : Ac-IETD-pNA, 9 : AC-LEHD-pNA). The amount of p-nitroaniline released by caspase activity was quantitated by measuring the optical density at 405 nm.

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