The activity of caspase-3 was measured by Caspase-3 Colorimetric Protease Assay (Life Technologies). Briefly, cells were resuspended in 50 μl lysis buffer and incubated on ice for 10 min. Cellular proteins (100 μg) were diluted in 50 μl lysis buffer and 50 μl reaction buffer (containing 10 mmol/L DTT). Five microliters of the 4 mmol/l DEVD-pNA substrate (200 μmol/l final concentration) were added, and the reaction was carried out at 37°C for 2 h in the dark. Reactions were measured in a microplate reader at 405 nm (A405).
Caspase 3 colorimetric protease assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Caspase-3 Colorimetric Protease Assay is a laboratory instrument designed to measure the activity of the caspase-3 enzyme, which is a key mediator of apoptosis or programmed cell death. The assay uses a colorimetric substrate that, when cleaved by active caspase-3, produces a colored product that can be detected and quantified using a spectrophotometer or plate reader.
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2 protocols using caspase 3 colorimetric protease assay
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Caspase-3 Activity Measurement Protocol
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Colorimetric Caspase-3 Activity Assay
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The activity of caspase 3 was measured by Caspase 3 Colorimetric Protease Assay (Life Technologies, Carlsbad, CA, USA). Cells were counted and pelleted (3–5 × 106 cells per sample). Cells were lysed with 50 μl of lysis buffer followed by protein quantification by the BCA method. Each cytosol extract was diluted to a concentration of 50–200 μg of protein per 50 μl of cell lysis buffer (1–4 mg/ml). Then 50 μl of 2× reaction buffer (containing 10 mM DTT) was added to each sample; 5 μl of the 4 mM DEVD-pNA substrate (200 μM final concentration) was added and incubated at 37°C for 2 h in the dark. Reactions were measured in a microplate reader at 405-nm wavelength.
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