Ubiquitylation Assay Kit

Product details

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 50uL 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

Other Notes
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.