This kit provides a simple and convenient assay for measuring the activity of caspase-1 and related caspases that recognize the sequence YVAD. The assay is based on the spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the substrate YVAD-pNA. pNA can be measured using a spectrophotometer or a microtiter plate reader at ~405 nm.
Catalogue Number
218790
Brand Family
Calbiochem®
References
Product Information
Detection method
Colorimetric
Form
100 Tests
Format
Cuvette or 96-well plate
Kit contains
Cell Lysis Buffer, Reaction Buffer, YVAD-pNA Substrate, DTT, Dilution Buffer, and a user protocol.
Irritating to eyes and skin. Risk of serious damage to eyes. Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
S Phrase
S: 26-36/39-24-61
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing and eye/face protection. Avoid contact with skin. Avoid release to the environment. Refer to special instructions/safety data sheet.
Product Usage Statements
Storage and Shipping Information
Ship Code
Ambient Temperature Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Storage
-20°C
Storage Conditions
Upon arrival store the entire contents of the kit at (-20°C). Store Cell Lysis Buffer, 2X Reaction Buffer, and Dilution Buffer at 4°C after thawing.
Protect from Light
Protect from light
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Cell Lysis Buffer, Reaction Buffer, YVAD-pNA Substrate, DTT, Dilution Buffer, and a user protocol.
Specifications
Documentation
Caspase-1 Assay Kit, Colorimetric Certificates of Analysis
Upon arrival store the entire contents of the kit at (-20°C). Store Cell Lysis Buffer, 2X Reaction Buffer, and Dilution Buffer at 4°C after thawing.
Principles of the assay
The Caspase-1 Colorimetric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the YVAD sequence. The assay is based upon spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the YVAD-pNA substrate. This cleavage can be quantified using a spectrophotometer or a microtiter plate reader at 400 or 405 nm. Comparing the absorbance of pNA from a treated sample with an untreated control allows determination of the increase in caspase-1 activity.
• Aliquot enough 2X reaction buffer for the number of assays to be performed. Add DTT to the 2X reaction buffer immediately before use (final concentration of 10 mM: Add 10 µl of 1.0 M DTT stock per 1 ml of 2X reaction buffer).
Detailed protocol
1. Induce apoptosis in cells by desired methods. Concurrently, incubate a control culture without any treatment. NOTE: Cytosolic extracts prepared from the human monocytic leukemia cell line THP-1 (ATCC #TIB-202) may be used as a source of active caspase-1 (positive control). The extract can be pre-activated by adding 10 mM DTT and incubating at 37°C for 1 h before use. 2. Count cells and pellet 2-5 x 106 cells. 3. Resuspend in 50 µl of chilled Cell Lysis Buffer and incubate on ice for 10 min. 4. Centrifuge for 1 min in a microcentrifuge (10,000 x g). 5. Transfer supernatant (cytosolic extract) to a fresh tube and keep on ice. 6. Determine the protein concentration. 7. Dilute 100-200 µg protein in 50 µl Cell Lysis Buffer for each assay. 8. Add 50 µl of 2X Reaction Buffer (containing 10 mM DTT) to each sample. 9. Add 5 µl of the 4 mM YVAD-pNA Substrate (200 µM final concentration) and incubate at 37°C for 1-2 h. 10. Read samples at 400 nm or 405 nm in a plate reader, or spectrophotometer using a 100 µl micro quartz cuvette, or dilute sample to 1 ml with dilution buffer and use a non-quartz cuvette. NOTE: Dilution of the samples proportionally decreases the absorbance. You may also perform the entire assay directly in a 96-well plate. Any increase in caspase-1 activity can be determined by comparing these results with the level of the untreated control. NOTE: Background reading from cell lysates and buffers should be subtracted from the readings of both treated and the untreated samples before calculating any increase in caspase-1 activity.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
