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662200 Ubiquitinated Protein Enrichment Kit

Ubiquitinated Protein Enrichment Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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      Overview

      Replacement Information
      Description
      OverviewA rapid method for isolating ubiquitinated proteins using affinity beads comprised of a GST-fusion protein containing an ubiquitin-associated sequence bound to glutathione-agarose. Useful for the enrichment of polyubiquitinated proteins from cell and tissue lysates of a broad range of species including canine, human, mouse, and yeast. The ubiquitinated proteins can be identified by loading the beads directly onto SDS-PAGE and then immunoblotting with the antibody of choice or Anti-Ubiquitin (Cat. No. 662099). Alternatively, it is possible that the beads can be treated with Isopeptidase T (Cat. No. 419700) to release the proteins from the ubiquitin chains.
      Catalogue Number662200
      Brand Family Calbiochem®
      References
      ReferencesChen, L. and Madura, K. 2002. Mol. Cell Biol. 22, 4902.
      Chen, L., et al. 2001. EMBO Rep. 2, 933.
      Product Information
      DeclarationSold under license of PCT Application wo 03/049,602.
      Form1 kit sufficient to process 12.5-25 mg lysate
      Kit containsPolyubiquitin Affinity Beads, Control Glutathione-Agarose Beads, Control Lysate, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 23/24-28-32-42-50/53

      Toxic by inhalation and in contact with skin.
      Very toxic if swallowed.
      Contact with acids liberates very toxic gas.
      May cause sensitization by inhalation.
      Very toxic to aquatic organisms; may cause long-term adverse effects in the aquatic environment.
      S PhraseS: 26-27-28-36/37/39-45-60-61

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Take off immediately all contaminated clothing.

      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      This material and its container must be disposed of as hazardous waste.
      Avoid release to the environment. Refer to special instructions/safety data sheet.
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store entire contents of the kit at 4°C.
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsPolyubiquitin Affinity Beads, Control Glutathione-Agarose Beads, Control Lysate, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      662200 0

      Documentation

      Ubiquitinated Protein Enrichment Kit SDS

      Title

      Safety Data Sheet (SDS) 

      Ubiquitinated Protein Enrichment Kit Certificates of Analysis

      TitleLot Number
      662200

      References

      Reference overview
      Chen, L. and Madura, K. 2002. Mol. Cell Biol. 22, 4902.
      Chen, L., et al. 2001. EMBO Rep. 2, 933.

      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
    • Naoto Inukai, et al. (2004) A novel hydrogen peroxide-induced phosphorylation and ubiquitination pathway leading to RNA polymerase II proteolysis. Journal of Biological Chemistry 279, 8190-8195.
    • Zhigang Zhang, Jin-Ying Wu, William Hait and Jin-Ming Yang. (2004) Regulation of the stability of P-glycoprotein by ubiquitination.. Molecular Pharmacology 66, 395-403.
      		•
      User Protocol

      Revision31-August-2010 RFH
      Form1 kit sufficient to process 12.5-25 mg lysate
      StorageUpon arrival store entire contents of the kit at 4°C.
      BackgroundA vast majority of short-lived proteins are degraded by the ubiquitin-proteasome pathway. A protein marked for degradation is covalently attached to multiple molecules of ubiquitin, a highly conserved 76-amino acid (8.6 kDa) protein, by a multi-enzymatic system consisting of Ubiquitin-activating (E1), Ubiquitin-conjugating (E2), and the Ubiquitin-ligating (E3) enzymes. The E1 activates a Ubiquitin monomer at its C-terminal cysteine residue to a high-energy thiolester bond which is then transferred to a reactive cysteine residue of the E2 enzyme. The final transfer of ubiquitin to e-amino group of a reactive lysine residue of substrate proteins is brought about by the E3 enzyme. Ubiquitinated protein is then escorted to the 26S proteasome where it undergoes final degradation and the ubiquitin is released and recycled. A family of proteins including Rad23, contain two ubiquitin-associated domains that bind ubiquitinated cellular proteins and translocate them to the proteasome. Ubiquitinated proteins can be enriched using affinity beads comprised of a GST-fusion protein containing this ubiquitin-associated sequence conjugated to glutathione-agarose.
      Principles of the assayThis kit is useful for the enrichment of polyubiquitinated proteins from cell and tissue lysates of a broad range of species including, canine, human, mouse, and yeast (see figure below). The ubiquitinated proteins can be identified by loading the beads directly onto SDS-PAGE and then immunoblotting with the antibody of choice or Anti-Ubiquitin (Cat. No. 662099). Alternatively, it is possible that the beads can first be treated with Isopeptidase T (Cat. No. 419700) to release the proteins from the ubiquitin chains.

      Figure 1: Principle of the Assay
      Materials providedQuantities sufficient for 25 affinity purification assays

      • Polyubiquitin Affinity Beads (Kit Component No. KP30801): Suspension of affinity beads in PBS containing 0.05% sodium azide. 1.0 ml.
      • Control Beads (Kit Component No. KP30802): Suspension of control beads in PBS containg 0.05% sodium azide. 200 µl.
      • Control Lysate (Kit Component No. KP30803): Suspension of protein extract in 1.5 M Tris-HCl, 10% glycerol, 5% β-mercaptoethanol, 2% SDS, 0.02% bromophenol blue, pH 6.8. 60 µl.
      Detailed protocol1. Resuspend the matrix by gentle inversion, until the beads are completely unpacked.
      2. Use a large bore pipet tip (by cutting off the terminal 3 mm with a razor blade) to remove ~40 µl of the affinity bead suspension. There is no need to wash the beads.
      3. Add the beads directly to 0.5 mg-1.0 mg of cell lysate, at a concentration of ~1mg/ml. The protein sample should be clear of any sediments or particulate matter, since this material will be recovered with the beads through subsequent washes.

      *Cell lysates can be prepared using a lysis buffer (pre-cooled to 4°C) consisting of: 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl and 1% Triton® X-100 detergent. Add a pre-made protease inhibitor cocktail (eg. Cat. No. 539134) and 10 mM N-ethylmaleimide (prepared in DMSO) immediately before use. Cells can be further disrupted by sonication, hydrostatic pressure, glass-bead agitation, or with osmotic destabilizers.

      4. Incubate at 4°C for 2-4 h with constant mixing to keep the affinity beads well suspended. Avoid aeration or vigorous mixing.
      5. Remove supernatant after centrifugation for 5 s at 4°C in a microfuge (~1000 X g), and resuspend in 1 ml of Wash buffer pre-cooled to 4°C (Wash buffer is the same as lysis buffer in step 3 without NEM and protease inhibitors). Repeat three more times.
      6. Suspend the affinity matrix in 40 µl of 2X gel loading buffer (gel loading buffer should be at room temperature and consist of 250 mM Tris, HCl, pH 6.8; 4% SDS; 10% β-mercaptoethanol; 20% glycerol; and bromophenol blue), and boil for 5 min.
      7. Centrifuge the material for 1 min at full speed in a microfuge (> 10,000 X g), and apply the supernatant to an 8-12% SDS-PAGE.
      8. For immunoblotting analysis, transfer the resolved proteins to nitrocellulose (preferably 0.2 mm), and stain with Ponceau S to confirm efficiency of transfer.

      To determine if a protein of interest is ubiquitinated:

      9. Wash the immunoblot 3 times with 1X TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% TWEEN®-20 detergent), using 100 ml per wash for 10 min each.
      10. Block the nitrocellulose membrane for 30 min on a rocking platform with TBST 5% milk solution for monoclonals and 10% for polyclonals at room temperature using about 1 ml per cm2 of membrane.
      11. Wash the nitrocellulose membrane 4 times in TBST for 10 min each on a rocking platform. Incubate the nitrocellulose membrane for 60 minutes on a rocking platform with primary antibody diluted in TBST, 5% milk.
      12. Wash the nitrocellulose membrane 4 times in TBST for 10 min each on a rocking platform.
      13. Incubate with secondary antibody enzyme conjugate diluted in 5% milk TBST for 60 min on a rocking platform.
      14. Wash the nitrocellulose membrane 4 times in TBST for 10 min each on a rocking platform.
      15. Develop with enhanced chemiluminescence to maximize detection.

      To confirm that ubiquitinated proteins are purified:

      16. Immerse the nitrocellulose filter in 1 cm of distilled water and boil for 5 minutes (The boiling step should only be used for detecting ubiquitin). To prevent the filter from floating, place a glass plate on top of the filter while boiling. Carefully remove the filter with forceps and immediately place in a tray containing 5% milk powder in 1X TBST. Incubate for 1 h at ambient temperature with agitation.
      18. Wash the filter three times with 1X TBST (100 ml per wash) for 10 min.
      19. Transfer the filter to a clean container and incubate with antibody against ubiquitin. Follow steps 12-15 from above.
      20. Develop with enhanced chemiluminescence to maximize detection.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Triton® is a registered trademark of Dow Chemical Company
      Tween® is a registered trademark of ICI Americas, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.