Key features and details
- Assay type: Enzyme activity
- Sample type: Cell Lysate, Purified protein
Product nameUbiquitylation Assay Kit (HeLa lysate-based)
See all Ubiquitylation kits
Sample typeCell Lysate, Purified protein
Assay typeEnzyme activity
Abcam Ubiquitylation Assay Kit (HeLa lysate-based) (ab139471) facilitates controlled ubiquitin conjugation of substrate proteins (exogenous or endogenous) of interest through the ubiquitin cascade. Conjugate formation can be detected and monitored by Western blotting using the highly sensitive ubiquitin-conjugate specific antibody supplied and/or antibodies for specific target proteins. Modified proteins can be subjected to further purification prior to their use in subsequent experiments if required.
Suggested uses for this kit include:
1) Generation of ubiquitin conjugated proteins.
2) Exogenous or endogenous HeLa lysate proteins (tagged/radio-labeled/immuno-detectable) can be ubiquitinylated followed by immediate detection/analysis.
3) Subsequent analysis could include proteasomal degradation, ubiquitin modification site mapping (by mass spectrometry), and the effect of ubiquitin modification on enzyme interactions, activity and function, Ubiquitinylation of proteins of interest from cell or tissue extracts.
4) Modification of proteins using ubiquitin derivatives or ubiquitin mutants for improved detection, analysis or investigation of alternative (non-proteasomal) ubiquitin signaling pathways.
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 ATP Regeneration Solution 1 x 100µl 10X Ubiquitin 2 x 50µl 10X Ubiquitinylation Buffer 1 x 100µl HeLa S100 Fraction 2 x 100µl Ubiquitin Aldehyde 1 x 50µl Ubiquitin Antibody Solution (mAb) 1 x 10µl
Ubiquitin-protein conjugate formation was detected by Western blotting of assays for A: General ubiquitinylation of endogenous HeLa S100 lysate proteins using the supplied Ubiquitin-protein Conjugates, pAb or B: specific modification of p53 present in HeLa S100 lysate using p53 specific monoclonal antibody.
Results demonstrate the utility of the Ubiquitylation Assay Kit (HeLa lysate-based) for both the ubiquitin modification of endogenousHeLa S100 lysate proteins in general and of specific endogenous proteins of interest, such as p53. The elevated level (A) or formation (B) of ubiquitin modified proteins can be clearly seen in the +ve (ATP containing) assays. The lower level (A) or absence (B) of ubiquitin conjugated proteins in –ve control reactions (-ATP) demonstrates that their formation is ATP-dependent (required for E1 activation) and, hence, derived from the ubiquitin cascade.
ab139471 has not yet been referenced specifically in any publications.