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539159 26S Proteasome Degradation Kit

26S Proteasome Degradation Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Radiometric
      Description
      Overview

      This product has been discontinued.





      Useful in the degradation of pre-formed ubiquitin-protein conjugates by the 26S proteasome.
      Catalogue Number539159
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Ubiquitin-protein conjugates (5000-15,000 cpm; 50-100 nM)
      • 10% TCA
      • 5% bovine serum albumin (BSA)
      References
      ReferencesGanoth, D., et al. 1988. J. Biol. Chem. 263, 12412.
      Hershko, A., et al. 1986. J. Biol. Chem. 261, 11992.
      Hershko, A., et al. 1984. Proc. Natl. Acad. Sci. USA 81, 1619.
      Hershko, A., et al. 1983. J. Biol. Chem. 258, 8206.
      Product Information
      Detection methodRadiometric
      Kit containsA partially purified Protein Fraction containing 26S Proteasome from Rabbit Cells, ATP Solution, Energy Solution, and a user protocol. Does not contain the conjugation enzymes (E1, E2s, or E3s).
      Applications
      Biological Information
      Assay time1 h
      Sample TypeUbiquitin-protein conjugates
      Physicochemical Information
      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® 26S Proteasome Degradation Kit is intended for degradation of pre-formed ubiquitin-protein conjugates in vitro.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Standard Handling
      Storage ≤ -70°C
      Storage ConditionsUpon arrival store the entire contents of the kit at -70°C. Once thawed, keep on ice prior to use. Following initial thaw, aliquot and freeze (-70°C).
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsA partially purified Protein Fraction containing 26S Proteasome from Rabbit Cells, ATP Solution, Energy Solution, and a user protocol. Does not contain the conjugation enzymes (E1, E2s, or E3s).
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      539159 0

      Documentation

      References

      Reference overview
      Ganoth, D., et al. 1988. J. Biol. Chem. 263, 12412.
      Hershko, A., et al. 1986. J. Biol. Chem. 261, 11992.
      Hershko, A., et al. 1984. Proc. Natl. Acad. Sci. USA 81, 1619.
      Hershko, A., et al. 1983. J. Biol. Chem. 258, 8206.

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Proteasome/Ubiquitination NF-kB Pathway Brochure

      Citations

      Title
    • Xiaotang Hu, et al. (2002) Ubiquitin/proteasome-dependent degradation of D-type cyclins is linked to tumor necrosis factor-induced cell cycle arrest.. Journal of Biological Chemistry 277, 16528-16537.
    • Peter Peters, et al. (2002) Arfaptin 2 regulates the aggregation of mutant huntingtin protein.. Nature Cell Biology 4, 240-245.
      		•
      User Protocol

      Revision16-September-2010 RFH
      Detection methodRadiometric
      StorageUpon arrival store the entire contents of the kit at -70°C. Once thawed, keep on ice prior to use. Following initial thaw, aliquot and freeze (-70°C).
      Intended useThe Calbiochem® 26S Proteasome Degradation Kit is intended for degradation of pre-formed ubiquitin-protein conjugates in vitro.
      Principles of the assayA series of specialized chromatographic steps is used to yield a partially purified protein fraction containing 26S proteasome without contaminating conjugation enzymes (E1, E2s, E3s). This stable fraction is preferred for the degradation of pre-formed ubiquitin-protein conjugates by the 26S proteasome since highly purified 26S is costly and labile. Radioactive ubiquitin-protein conjugates are mixed with the 26S proteasome fraction and an energy solution containing ATP and Mg2+ and allowed to incubate. At desired time points aliquots are removed, precipitated with TCA, and the TCA-soluble fraction counted using a scintillation counter. The TCA-soluble counts are plotted against time. Degradation is indicated as an increase in TCA-soluble counts over time.
      Materials provided• 26S Proteasome Fraction (Kit Component No. KP8701): 1 vial, 50 µl, in 500 mM Mg2+/ATP, 150 mM NaCl, 50 mM HEPES, 10% glycerol, pH 7.6; please refer to vial label for lot-specific concentration
      • Energy Solution (Kit Component No. KP8702): 1 vial, 75 µl, supplied as a 10X solution of 20 mM Mg2+/ATP, pH 7.0
      Materials Required but not provided• Ubiquitin-protein conjugates (5000-15,000 cpm; 50-100 nM)
      • 10% TCA
      • 5% bovine serum albumin (BSA)
      Detailed protocol1. Combine ubiquitin-protein conjugates, 5 µl Energy Solution, and 26S Proteasome Fraction (5 to 10 nM final concentration) in a microfuge tube; add buffer to a final volume of 50 µl.
      2. Incubate at 37°C for 0-90 min.
      3. At desired time points, remove 10 µl of the reaction and add to 600 µl 10% TCA. Mix and add 200 µl 5% BSA as a carrier.
      4. Vortex and place on ice for 15 min.
      5. Centrifuge at 4°C for 10 min in a microcentrifuge at 14,000 rpm.
      6. Add 650 µl of the supernatant to the counting vial containing scintillation liquid and count.
      7. Plot the increase in TCA-soluble counts against time.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.