Product nameUbiquitylation Assay Kit
See all Ubiquitylation kits
Sample typePurified protein
Ubiquitylation Assay Kit (ab139467) provides the means of generating thioeseter linked, activated ubiquitin-E1 conjugates, utilizing the first step in the ubiquitin cascade, for investigation of ubiquitin activation, subsequent ubiquitin transfer to/interaction with E2 conjugating enzymes and their use in the ubiquitinylation of E3 ligases and target substrate proteins.
The reagents supplied are intended to be used in conjunction with user supplied wild type or mutant E2 enzymes in E1 initiated/mediated reactions. Kit is supplied with a highly sensitive ubiquitin antibody for detection of ubiquitin and ubiquitin conjugates. Kit provides sufficient material for 20 x 50µL reactions.
Suggested uses for this kit include:
1) Activation of ubiquitin for conjugation to wild type or mutant E2 enzymes (user supplied) via thioester bond formation.
2) Use of Ub-E2 conjugates produced in the subsequent ubiquitin modification of specific target proteins in presence of a dedicated ubiquitin E3 ligase.
3) Investigation of ubiquitin activation by E1 activating enzyme.
4) Substitution of the wild type ubiquitin provided with ubiquitin mutants or derivatives (e.g. biotinylated-ubiquitin) allowing their activation and subsequent utility in the ubiquitin cascade.
Note: Protocols provided for applications 1 and 3. Assay set-up can be readily modified for alternative applications by inclusion, omission or substitution of specific enzyme components
The covalent attachment of ubiquitin to proteins (ubiquitinylation) and their subsequent proteasomal degradation plays a fundamental role in the regulation of cellular function through biological events involving cell cycle, differentiation, immune responses, DNA repair, chromatin structure, and apoptosis.
Ubiquitinylation 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.
Storage instructionsPlease refer to protocols.
Components 20 tests 10X Ubiquitinylation Buffer 1 x 100µl 20X Mg-ATP Solution 1 x 50µl 20X Ubiquitin Activating Enzyme Solution (E1) 1 x 50µl 20X Ubiquitin Solution (Ub) 1 x 50µl 2X Non-reducing Gel Loading Buffer 1 x 1.25ml Ubiquitin Antibody Solution 1 x 25µl
Western blot of ubiquitin thioester assays for the E1 activating enzyme provided and an example E2, UbcH5a (user supplied). Procedures as described in “Assay Protocol” section. Ubiquitin-enzyme conjugates were detected by Western blotting of E1-Ub (A/B) or E2-Ub (C/D) thioester assays in the presence (A/C) or absence (B/D) of Mg-ATP co-factor, using the Ubiquitin antibody provided, as described in “Western Blot Analysis” section. Results demonstrate the formation of ubiquitin thioester linked E1 and/or E2 conjugates in all +Mg-ATP reactions. The absence of such conjugates in negative control reactions demonstrates that their formation is Mg-ATP dependent (required for E1 activation) and hence derived from the ubiquitin cascade.