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539176 Proteasome Isolation Kit, Human

Proteasome Isolation Kit, Human MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
539176
  
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      Overview

      Description
      Overview

      This product has been discontinued.



      A rapid method for the isolation of biologically active proteasomes using affinity matrix beads comprised of a GST-fusion protein containing an ubiquitin-like domain (UbL) bound to GST-agarose. The proteasome subunit proteins can be identified by loading the beads directly onto an SDS-PAGE gel and immunoblotting with subunit specific antibodies. Alternatively, proteasome bound to beads can be used in proteolytic assays using proteasome substrates.

      • Utilize Ubiquitin-like domain (Ubl) bound to GST-agarose
      • Rapidly isolate biologically active proteasomes
      • Affinity mature beads can be directly loaded onto SDS-PAGE gels for proteasome subunit identification
      • Proteasome-bound beads are compatible with proteolytic assays using proteasome substrates

      Catalogue Number539176
      Brand Family Calbiochem®
      Application Data
      Total protein extracts were applied to control and proteasome-binding beads, and incubated for 4 h at 4°C. The matrix was washed three times with Buffer A, suspended in SDS sample buffer, separated in a 10% SDS-polyacrylamide gel, and electroblotted to nitrocellulose. The filter was incubated with antibodies against Rpt1, a subunit in the 19S regulatory particle of the proteasome. The first lane shows the recovery of the proteasome subunit Rpt1 with the proteasome-binding GST-UbL beads. The second lane is a mock-reaction (using GST), while the third lane contains 5% of the input lysate. Similar samples were incubated in reaction buffer to measure proteasome-specific proteolytic activity. The hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC was readily detected in the GST-UbL beads, while the no activity was observed with the GST beads.
      Materials Required but Not Delivered• Lysis buffer: 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl and 1% Triton® X-100 detergent. Addition of 2 mM ATP to the lysate will improve the recovery of intact 26S proteasome.
      • Wash Buffer (Buffer A): Lysis buffer with protease inhibitors such as Antipain, aprotinin, chymostatin, leupeptin, and pepstatin. Alternatively, a pre-made protease inhibitor cocktail such as Cat. No. 539134 can be used.
      • Reaction Buffer: 25 mM Hepes, 500 mM EDTA, pH 7.6
      • Gel Loading Buffer: 250 mM Tris, HCl, pH 6.8; 4% SDS; 10% β-mercaptoethanol; 20% glycerol; and bromophenol blue
      • TBST: 10 mM Tris-HCl, 150 mM NaCl, 0.1% TWEEN®-20 detergent
      References
      ReferencesChen, L., and Madura, K. 2002. Mol. Cell Biol. 22, 4902.
      Chen, L., et al. 2001. EMBO Rep. 2, 933.
      Schauber, C., et al. 1998. Nature 391, 715.
      Product Information
      DeclarationSold under license of U.S. Patent 6,294,363.
      Format1.0 ml bead suspension
      Kit containsUbL Beads Sufficient to process 12.5 to 25 mg of Lysate, Control Glutathione-Agarose Beads, Control Lysate, and a user protocol.
      Quality LevelMQ100
      Biological Information
      Assay time3-5 h
      Sample TypeCell extracts
      Species Reactivity
      • Canine
      • Human
      • Mouse
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store entire kit contents at 4°C.
      Do not freeze Yes
      Supplemental Information
      Kit containsUbL Beads Sufficient to process 12.5 to 25 mg of Lysate, Control Glutathione-Agarose Beads, Control Lysate, and a user protocol.
      Global Trade Item Number
      Catalogue Number GTIN
      539176 0