E2-Ubiquitin Conjugation Kit (ab139472)
Key features and details
- Assay type: Enzyme activity
- Sample type: Purified protein
Overview
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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).
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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
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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
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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
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Western Blot of Thioester Assays (TE +ve/-ve controls) for all E2 conjugating enzymes provided
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.
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Western blot analysis of Thioester assays (TE (+)ve/(-)ve control)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).
