Millipore Sigma Vibrant Logo
Attention: We have moved. Merck Millipore products are no longer available for purchase on MerckMillipore.com.Learn More

662096 Ubiquitin Protein Conjugation Kit

Ubiquitin Protein Conjugation Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
662096
  
Purchase on Sigma-Aldrich

Overview

Replacement Information
Description
Overview

This product has been discontinued.





Useful for the formation of ubiquitinated proteins that are labeled or immuno-detectable. Has been shown to work with typical iodinated substrate proteins such as lysozyme and β-lactoglobulin. Addition of Ubiquitin Aldehyde (Cat. No. 662056) is recommended for the inhibition of ubiquitin C-terminal hydrolase and to improve conjugate yield. Fractions do not contain 20S, 26S, or any other conjugate or protein degradation activity. Each kit includes two enzyme fractions, ubiquitin, ATP solution, and a user protocol.
Catalogue Number662096
Brand Family Calbiochem®
References
Product Information
Kit containsConjugation Fraction #1, Conjugation Fraction #2, Ubiquitin, Energy Solution, and a user protocol.
Applications
Biological Information
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Intended useThe Calbiochem® Ubiquitin Protein Conjugation Kit is intended for the in vitro preparation of ubiquitinated proteins that are labeled or can be used for immunodetection.
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. After thawing, keep on ice prior to use. For best results, aliquot and freeze. AVOID REPEATED FREEZE/THAW CYCLES.
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsConjugation Fraction #1, Conjugation Fraction #2, Ubiquitin, Energy Solution, and a user protocol.
Specifications
Global Trade Item Number
Catalogue Number GTIN
662096 0

Documentation

Ubiquitin Protein Conjugation Kit Certificates of Analysis

TitleLot Number
662096

Brochure

Title
Caspases and other Apoptosis Related Tools Brochure
Proteasome/Ubiquitination NF-kB Pathway Brochure

Citations

Title
  • Shamoto-Nagai, M., et al. 2004. Journal of Neural Transmission 111, 1253.
  • Hu, X., et al. 2002. Journal of Biological Chemistry 277, 16528.
  • Peters, P., et al. (2002) Nature Cell Biology 4, 240.
    		•
    User Protocol

    Revision25-June-2008 JSW
    StorageUpon arrival, store the entire contents of the kit at -70°C. After thawing, keep on ice prior to use. For best results, aliquot and freeze. AVOID REPEATED FREEZE/THAW CYCLES.
    Intended useThe Calbiochem® Ubiquitin Protein Conjugation Kit is intended for the in vitro preparation of ubiquitinated proteins that are labeled or can be used for immunodetection.
    Principles of the assayThe Calbiochem® Ubiquitin Protein Conjugation Kit is supplied with the full compliment of purified conjugation enzymes (E1, E2s, and E3s) that are found in Fraction II. The enzymes have been tested and shown to work with typical iodinated "n-end rule" substrates proteins such as lysozyme and β-lactoglobulin. These conjugation fractions contain ubiquitin C-terminal hydrolases. The addition of Ubiquitin Aldehyde (Cat. No. 662056) is recommended for the inhibition of UCHs and to improve overall conjugate yield. The supplied fractions do not contain 20S, 26S, or any other conjugate or protein degradation activity.
    Materials provided• Conjugation Fraction #1 (Kit Component No. KP8801): 1 vial, 125 µl, supplied at 800 µg/ml
    • Conjugation Fraction #2 (Kit Component No. KP8802): 1 vial, 125 µl, supplied at 800 µg/ml
    • Ubiquitin (Kit Component No. KP8803): 1 vial, 25 µl, supplied at 20 mg/ml (2.35 mM) in 150 mM NaCl, 50 mM HEPES, 500 µM DTT, pH 7.6.
    • Energy Solution (Kit Component No. KP8804): 1 vial, 75 µl, supplied as a 10X concentrated solution
    Detailed protocolNote: Individual results may vary with different conditions and substrates.

    1. Combine assay components as follows:

    Table 1: Assay Components


    2. If Ubiquitin Aldehyde is being used, pre-incubate for 5 min at 37°C to allow for inhibition of UCHs.
    3. Initiate conjugation reaction by addition of radiolabeled substrate. Mix by vortexing gently.
    4. Incubate 0.5-4 h at 37°C, depending on individual conditions and requirements. Taking 0.5-1 h time points during the first experiment is recommended for determination of optimal incubation times. Quench time points with EDTA, 10 mM final concentration. Note: DO NOT use these quenched samples for degradation assay.
    5. Run SDS-PAGE gel to determine conjugate formation. Identify optimal conditions.
    6. Once optimal conditions have been determined, the reaction mixture may be aliquoted and frozen for future use, or if desired, conjugates can be further purified using a spin column (G-50). DO NOT quench with EDTA.
    7. Final samples should be counted and run on a gel. Appropriate aliquots should be made and frozen at -80°C. The addition of a co-solvent (PEG, Sucrose or Glycerol) is optional.
    Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
    Interactive Pathways™ is a trademark of EMD Chemicals, Inc.