Description

PRINCIPLE OF THE ASSAY

Caspase 3 and caspase 7 share similar substrate specificity by recognizing tetra-peptide motif Asp-x-x-Asp.[12] The C-terminal Asp is absolutely required while variations at other three positions can be tolerated.[13] Caspase-3 and 7 cleave a variety of cellular substrates after aspartic acid residues-a characteristic that is central to their role in mammalian apoptosis. The Caspase-3/7 Colormetric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the tetrapeptide sequence DEVD. The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (p-NA) after cleavage from the p-NA-labeled Ac-DEVD-pNA. The p-NA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400-410nm. Comparison of the absorbance of p-NA from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase-3/7 activities.

Assay Procedure
1. Treat cells by desired method. We recommend conducting four reactions (1) non-induced cells, (2) non-induced cells + inhibitor, (3) induced cells, and (4) induced cells + inhibitor as well as two control reactions (1) caspase 3/7 positive control, and (2) caspase 3/7 positive control + inhibitor.
2. Count cells and pellet 1 x 106 cells per test in 1.5 ml tube.
3. Resuspend cells in 100 µl of chilled 1 x Assay Buffer and incubate cells on ice for 10 minutes.
4. Centrifuge at 10,000 x g for 1 min.
5. Transfer supernatant (cytosolic extract) to each well of 96-well plate (or a fresh tube if a spectrophotometer is later used) and put on ice for immediate assay (or store at -80C for future use).
6. Immediately before use, prepare sufficient working reagents per assay: 10 µl of Substrate, 5 µl of 200 mM DTT and 85 µl of 1 x Assay Buffer.
7. Add 100 µl of the Working Reagents prepared as above to each well of 96-well plate.
8. Seal the plate with plate sealer. Incubate at 37C for 1~2 hours and protect from light.
9. Read the pate at 405 nm (wavelength can range from 400 to 410 nm) in a microplate reader (or a spectrophotometer if microtubes are used).
10. Fold-increase in caspase-3/7 activities can be determined by comparing the OD405 with that of the uninduced control or other desired controls.

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