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QIA89 Glutathione Apoptosis Assay Kit

Glutathione Apoptosis Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
QIA89
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      Overview

      This product has been discontinued.





      A fluorometric assay to detect the in vitro changes in glutathione (GSH) levels during apoptosis. Utilizes monochlorobimane (MCB), a dye that fluoresces blue when bound to GSH. The adduct formation is catalyzed by glutathione S-transferase.
      Catalogue NumberQIA89
      Brand Family Calbiochem®
      Materials Required but Not Delivered•Fluorimeter or fluorescence plate reader that can detect fluorescence at an Excitation max = ~380 nm and Emission max = ~460 nm
      •PBS
      •Apoptosis inducer of choice
      References
      Product Information
      Detection methodFluorometric
      Form100 Tests
      Format96-well plate or cuvette
      Kit containsCell Lysis Buffer, Monochlorobimane Reagent, GST Reagent, and a user protocol.
      Applications
      Key Applications Fluorometric
      Biological Information
      Assay time1.5-2 h
      Sample TypeCell lysate
      Physicochemical Information
      Emission max.
      Excitation max.
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/37/38

      Irritating to eyes, respiratory system and skin.
      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
      Intended useThe Calbiochem® Glutathione Apoptosis Detection Kit is intended for the measurement of changes in the glutathione level in cell lysates.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsUpon arrival store the Monochlorobimane Reagent and GST Reagent at -20°C and the Cell Lysis Buffer at 4°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCell Lysis Buffer, Monochlorobimane Reagent, GST Reagent, and a user protocol.
      Specifications
      Global Trade ITEM Number
      Catalogue Number GTIN
      QIA89 0

      Documentation

      Glutathione Apoptosis Assay Kit Certificates of Analysis

      TitleLot Number
      QIA89

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      Kit SourceBook - 2nd Edition EURO
      User Protocol

      Revision06-Febuary-2009 RFH
      Form100 Tests
      Format96-well plate or cuvette
      Detection methodFluorometric
      StorageUpon arrival store the Monochlorobimane Reagent and GST Reagent at -20°C and the Cell Lysis Buffer at 4°C.
      Intended useThe Calbiochem® Glutathione Apoptosis Detection Kit is intended for the measurement of changes in the glutathione level in cell lysates.
      BackgroundGlutathione (GSH) is a low-molecular weight thiol that plays a critical role in the cellular defense against oxidative and nitrosative stress in mammalian cells. Diminished glutathione levels have been observed in the early stages of apoptosis.
      Principles of the assayThe Glutathione Apoptosis Detection Kit, Fluorometric utilizes monochlorobimane (MCB), a dye that forms an adduct exclusively with GSH. The unbound MCB is nearly nonfluorescent, whereas the dye fluoresces blue when bound to glutathione (Excitation max = ~380 nm; Emission max = ~460 nm). The reaction is catalyzed by glutathione-S-transferase. Thus, the changes in glutathione levels during apoptosis can be easily detected using a fluorometer or a 96-well fluorometric plate reader.
      Materials providedThere are sufficient reagents to perform 100 tests.

      • Cell Lysis Buffer (Kit Component No. JA5400-25ML): 1 bottle, 25 ml
      • Monochlorobimane Reagent (Kit Component No. JA5401-200UL): 1 vial, 200 µl, 25 mM in DMSO
      • GST Reagent (Kit Component No. JA5402-200UL): 1 vial, 200 µl, 50 U/ml
      • GSH standard (Kit Component No. JA5403-1MG): 1 vial, 1 mg
      Materials Required but not provided•Fluorimeter or fluorescence plate reader that can detect fluorescence at an Excitation max = ~380 nm and Emission max = ~460 nm
      •PBS
      •Apoptosis inducer of choice
      Detailed protocolNote: the following protocol has been optimized using Jurkat cells. Other cell types in which glutathione levels drop druing apoptosis may also be used, however, the assay may need to be optimized for other cell types.

      1. Induce apoptosis in cells by desired method. Concurrently incubate control cells that are uninduced.
      2. Collect 1 x 106 cells by centrifugation at 700 x g for 5 min at 4°C.
      3. Remove the supernatant and resuspend the cell pellet in 1 ml ice-cold PBS.
      4. Transfer to a 1.5 microcentrifuge tube and centrifuge at 700 x g for 5 min at 4°C. Remove the supernatant and discard.
      5. Resuspend the cell pellet in 100 µl ice-cold Cell Lysis Buffer.
      6. Incubate for 10 min on ice. Centrifuge in a microcentrifuge at 14,000 x g for 10 min.
      7. Transfer the supernatant to a clean tube and discard the pellet.
      8. Add 2 µl Monochlorobimane Reagent and 2 µl GST Reagent to each tube. Prepare a negative control sample by adding 1 µl Monochlorobimane Reagent to 100 µl Cell Lysis Buffer.
      9. Incubate all samples and controls at 37°C for 15-30 min.
      10. Measure the fluorescence using a fluorimeter or fluorescence plate reader with an excitation wavelength of ~380 nm and an emission wavelength of ~460 nm.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.