Caspase-9 Colorimetric Assay Kit

Antigen: Caspase 9, Apoptosis-Related Cysteine Peptidase (CASP9)

Synonyms: MCH6, APAF3, APAF-3, ICE-LAP6, CASPASE-9c, Mch6, AI115399, AW493809, Caspase-9, CASP9, LOC100101592

Application: Enzyme Activity Assay (EAA)

Catalog no.: ABIN411877

Additional Information

Alternative name: Caspase-9

Description: Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. The Caspase-9 Colorimetric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence LEHD. The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate LEHD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400- or 405-nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase-9 activity.

Application Details

Protocol: A. General Considerations Aliquot enough 2X Reaction Buffer for the number of assays to be performed. Add DTT to the 2X Reaction Buffer immediately before use (10 mM final concentration: add 10 μ l of 1.0 M DTT stock per 1 ml of 2X Reaction Buffer). Store kit at –20 o C (Store Cell Lysis Buffer, 2X Reaction Buffer, and Dilution Buffer at 4 o C after opening). All reagents are stable for 6 months under proper storage conditions. Protect LEHD-pNA from light. B.. 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Count cells and pellet 2-5 x 10 6 cells. 3. Resuspend cells in 50 μ l of chilled Cell Lysis Buffer and incubate cells on ice for 10 minutes. 4. Centrifuge for 1 min in a microcentrifuge (10,000 x g). 5. Transfer supernatant (cytosolic extract) to a fresh tube and put on ice. 6. Assay protein concentration. 7. Dilute 100-200 μ g protein to 50 μ l Cell Lysis Buffer for each assay. 8. Add 50 μ l of 2X Reaction Buffer (containing 10 mM DTT) to each sample. Add 5 μ l of the 4 mM LEHD-pNA substrate (200 μ M final conc.) and incubate at 37 o C for 1-2 hour. 9. Read samples at 400- or 405-nm in a microtiter plate reader, or spectrophotometer using a 100- μ l micro quartz cuvet (Sigma), or dilute sample to 1 ml with Dilution Buffer and using regular cuvet (note: Dilution of the samples proportionally decreases the reading). You may also perform the assay in a 96-well plate. Fold-increase in Caspase-9 activity can be determined by comparing the results of treated samples with the level of the uninduced control.

Application Notes: Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in Caspase-9 activity.

Restrictions: For Research Use only