Overview

  • Product name

    E2-Ubiquitin Conjugation Kit
  • Sample type

    Purified protein
  • Assay type

    Enzyme activity
  • Product overview

    E2-Ubiquitin Conjugation Kit (ab139472) provides the tools necessary for generating a range of thioester-linked ubiquitin-conjugated E2 enzymes for use in ubiquitinylation experiments. Biotinylated ubiquitin (provided in the kit) can be detected with streptavidin-linked enzymes via SDS PAGE and western blotting.


    This product provides sufficient material for a total of 50 reactions: 4 reactions with each included E2 enzyme.


    Uses for this kit include:


    1. Ubiquitinylation of target proteins in presence of dedicated E3 ligase. Panel of E2s provided for generation of E2-Ub thioester conjugates for testing vs. specific E3/target combinations. For example: ubiquitinylation of p53 in the presence of mdm2 (E3) and UbcH5b (E2).


    2. Activation of Ub for thioester conjugation to novel E2 enzymes (substituted like for like with kit E2s, under directly comparable conditions).

  • Notes

    Ubiquitinylation, the covalent attachment of ubiquitin to proteins, is achieved through three enzymatic steps. In an ATP-dependent process, the ubiquitin activating enzyme (E1) catalyzes the formation of a reactive thioester bond with ubiquitin, in the presence of a Mg2+ cofactor, followed by its subsequent transfer to the active site cysteine of a ubiquitin carrier protein (E2). The specificity of ubiquitin ligation arises from the subsequent association of the E2-ubiquitin thioester with a substrate specific ubiquitin-protein isopeptide ligase (E3), which facilitates the formation of the isopeptide linkage between ubiquitin and its target protein.

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components 50 tests
    10X Ubiquitinylation Buffer 1 x 250µl
    20x Biotinylated Ubiquitin Solution 1 x 125µl
    20X Mg-ATP Solution 1 x 125µl
    20X Ubiquitin Activating Enzyme Solution (E1) 1 x 125µl
    2X Non-reducing Gel Loading Buffer 2 x 1.25ml
    UbcH1 (Human, Recombinant) 1 x 20µl
    UbcH10 (Human, Recombinant) 1 x 20µl
    UbcH13/Mms2 (Human, Recombinant) 1 x 20µl
    UbcH2 (Human, Recombinant) 1 x 20µl
    UbcH3 (Human, Recombinant) 1 x 20µl
    UbcH5a (Human, Recombinant) 1 x 20µl
    UbcH5b (Human, Recombinant) 1 x 20µl
    UbcH5c (Human, Recombinant) 1 x 20µl
    UbcH6 (Human, Recombinant) 1 x 20µl
    UbcH7 (Human, Recombinant) 1 x 20µl
    UbcH8 (Human, Recombinant) 1 x 20µl
  • Research areas

  • Relevance

    Ubiquitin-conjugating enzymes, also known as E2 enzymes and more rarely as ubiquitin-carrier enzymes, perform the second step in the ubiquitination reaction that targets a protein for degradation via the proteasome.The ubiquitination process covalently attaches ubiquitin, a short protein of 76 amino acids, to a lysine residue on the target protein. Once a protein has been tagged with one ubiquitin molecule, additional rounds of ubiquitination form a polyubiquitin chain that is recognized by the proteasome's 19S regulatory particle, triggering the ATP-dependent unfolding of the target protein that allows passage into the proteasome's 20S core particle, where proteases degrade the target into short peptide fragments for recycling by the cell.

Images

  • Biotinylated-ubiquitin-enzyme conjugates were detected by Western Blotting on thioester assays containing UbcH1 (A), UbcH2 (B), UbcH3 (C), UbcH5a (D), UbcH5b (E), UbcH5c (F), UbcH6 (G), UbcH7 (H), UbcH8 (I), UbcH10 (J), Ubc13/MMS2 (K) respectively, using Streptavidin-HRP detection system. M:  Biotinylated SDS molecular weight marker, from bottom: 20.1, 29.0, 39.8, 58.1kDa.

  • Western blot analysis of Thioester assays (TE (+)ve/(-)ve control) for E2 conjugating enzyme UbcH2. Procedure as described in the assay set up section (without DTT), and ubiquitin conjugate detected by streptavidin AP. The absence of conjugate in TE (-)ve control control demonstrate that formation of TE is ATP dependent (required for E1 activation).

Protocols

References

This product has been referenced in:

  • Freitas FB  et al. African swine fever virus encodes for an E2-ubiquitin conjugating enzyme that is mono- and di-ubiquitinated and required for viral replication cycle. Sci Rep 8:3471 (2018). Read more (PubMed: 29472632) »
  • Lee JH  et al. Small heterodimer partner SHP mediates liver X receptor (LXR)-dependent suppression of inflammatory signaling by promoting LXR SUMOylation specifically in astrocytes. Sci Signal 9:ra78 (2016). Read more (PubMed: 27485016) »
See all 3 Publications for this product

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