218824 Caspase-9 Assay Kit, Colorimetric

218824
  
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      Overview

      Replacement Information
      Description
      Overview

      This product has been discontinued.





      This kit provides a simple and convenient assay for measuring the activity of caspase-9 and related caspases that recognize the sequence LEHD. The assay is based on the spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the substrate LEHD-pNA. pNA can be measured using a spectrophotometer or a microplate reader at ~405 nm.
      Catalogue Number218824
      Brand Family Calbiochem®
      References
      Product Information
      Detection methodColorimetric
      Form100 Tests
      FormatCuvette or 96-well plate
      Kit containsCell Lysis Buffer, Reaction Buffer, LEHD-pNA Substrate, DTT, Dilution Buffer, and a usher protocol.
      Applications
      Biological Information
      Assay time2-3 h
      Sample TypeCell lysate
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 22-36/37/38-44-41

      Harmful if swallowed.
      Irritating to eyes, respiratory system and skin.
      Risk of explosion if heated under confinement.
      Risk of serious damage to eyes.
      S PhraseS: 26-36

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      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, LEHD-pNA Substrate, DTT, Dilution Buffer, and a usher protocol.
      Specifications

      Documentation

      Caspase-9 Assay Kit, Colorimetric Certificates of Analysis

      TitleLot Number
      218824

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      User Protocol

      Revision18-October-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.
      BackgroundActivation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. The Caspase-9/Mch6 Colorimetric Protease Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the LEHD sequence. The assay is based upon spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the LEHD-pNA substrate. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400- or 405 nm. Comparing the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase-9 activity.
      Materials provided• Cell Lysis Buffer (Kit Component No. KP19201): 100 ml
      • 2X Reaction Buffer (Kit Component No. KP19202): 4 x 2 ml
      • LEHD-pNA Substrate (4 mM) (Kit Component No. KP19203): 500 µl
      • DTT (1 M) (Kit Component No. KP19204): 400 µl
      • Dilution Buffer (Kit Component No. KP19205): 100 ml
      Precautions and recommendations 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).
      After thawing, store the cell lysis buffer, 2X reaction buffer, and dilution buffer at 4°C.
      Protect LEHD-pNA substrate from light.
      Detailed protocol1. Induce apoptosis in cells by desired methods. Concurrently, incubate a control culture without any treatment.
      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. Assay 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 LEHD-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. Fold-increase in Caspase-9 activity can be determined by comparing these results with the level of the uninduced 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-9 activity.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.