Caspase Activity Assay Protocol

The activity of caspases was detected as previously described [58 (link)]. Cells were incubated with lysis buffer (R&D Systems, Minneapolis, MN, USA) for 10 min on ice. Total protein (100 μg) was incubated with 2X reaction buffer and a substrate of the colorimetric tetrapeptide, including DEVD-pNA for caspase-3, IETD-pNA for caspase-8, and LEHD-pNA for caspase-9. The reaction mixture was incubated at 37 °C for 2 h, and the enzyme-catalyzed release of p-nitroaniline was measured at 405 nm using a multi-well reader (Thermo Fisher Scientific).

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Kang Y.J., Jang J.Y., Kwon Y.H., Lee J.H., Lee S., Park Y., Jung Y.S., Im E., Moon H.R., Chung H.Y, & Kim N.D. (2022). MHY2245, a Sirtuin Inhibitor, Induces Cell Cycle Arrest and Apoptosis in HCT116 Human Colorectal Cancer Cells. International Journal of Molecular Sciences, 23(3), 1590.

Publication 2022

lysis buffer multi-well reader

Buffer Caspase 3 Caspase 8 Caspase 9 Caspases Cells Colorimetric Devd pna Enzyme Nitroaniline Protein

Corresponding Organization : Pusan National University

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Variable analysis

independent variables

Incubation with lysis buffer
Incubation with 2X reaction buffer and substrates (DEVD-pNA, IETD-pNA, LEHD-pNA)

dependent variables

Enzyme-catalyzed release of p-nitroaniline measured at 405 nm

control variables

Incubation time (10 min on ice for lysis, 2 h at 37 °C for reaction)
Total protein amount (100 μg)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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