218790 | Caspase-1 Assay Kit, Colorimetric

218790
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Colorimetric

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      218790-1KIT
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          Description
          OverviewThis 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 Number218790
          Brand Family Calbiochem®
          References
          Product Information
          Detection methodColorimetric
          Form100 Tests
          FormatCuvette or 96-well plate
          Kit containsCell Lysis Buffer, Reaction Buffer, YVAD-pNA Substrate, DTT, Dilution Buffer, and a user protocol.
          Applications
          Biological Information
          Assay time2.5-3 h
          Sample TypeCell lysates
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 36/38-41-52/53

          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 PhraseS: 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 ConditionsUpon 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 containsCell Lysis Buffer, Reaction Buffer, YVAD-pNA Substrate, DTT, Dilution Buffer, and a user protocol.
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          218790
          User Protocol

          Revision22-February-2010 RFH
          Form100 Tests
          FormatCuvette or 96-well plate
          Detection methodColorimetric
          StorageUpon 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 assayThe 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.
          Materials provided• Cell Lysis Buffer (Kit Component No. KP5701): 1 bottle, 100 ml
          • 2X Reaction Buffer (Kit Component No. KP5702): 4 vials, 2 ml each
          • YVAD-pNA Substrate (4 mM) (Kit Component No. KP5703): 1 vial, 500 µl; Protect from light
          • DTT (1 M) (Kit Component No. KP5704): 1 vial, 400 µl
          • Dilution Buffer (Kit Component No. KP5705): 1 vial, 100 ml
          Preparation 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 protocol1. 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 TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.