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Measurement of Activity of Caspase-3/9

XM Xiangchao Meng
HC Hongye Chen
ZT Zhihui Tan
WY Weitao Yan
YL Yinfeng Liu
JL Ji Lv
MH Meng Han
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The activity of caspase-3 and caspase-9 was assessed with Caspase 3 Activity Assay Kit or Caspase 9 Activity Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocols. Briefly, the cells were collected and lysed on the ice, and the protein concentration was determined with Bradford Protein Assay Kit (Beyotime Institute of Biotechnology). Afterwards, the sample was incubated with acetyl-Asp-Clu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) substrate at 37 ℃ for 1 h to produce the yellow pNA, and the absorbance at 405 nm was measured with a microplate reader. The standard curve was drawn using standard pNA with grading concentrations, and activity of caspase-3 or caspase 9 was calculated based on the pNA concentration. One unit was the amount of enzyme that will cleave 1.0 nmol of the colorimetric substrate Ac-DEVD-pNA per hour at 37 ℃ under saturated substrate concentrations.

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