Description
Key facts
Sample types:Purified protein, Cell Lysate,
Assay type:Enzyme activity
Product details:
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.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions--80°C, Appropriate long-term storage conditions--80°C, Storage information--80°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Ubiquitylation also called ubiquitination is a process where ubiquitin proteins attach to other proteins marking them for degradation or modifying their function. Ubiquitin is a small protein weighing about 8.6 kDa and it is highly expressed in eukaryotic cells. It tags substrates to regulate their turnover and activity involving a cascade of enzyme actions by E1 E2 and E3 ubiquitin ligases. The process is important for maintaining protein homeostasis and regulating many cellular processes.
Biological function summary
The ubiquitylation modifies protein function and stability. It is not a standalone process; ubiquitin often functions within large complexes such as the proteasome where tagged proteins are degraded. It controls the fate of proteins affecting processes like cell cycle DNA repair and signal transduction. An increase or decrease in ubiquitylation can affect protein levels within the HeLa lysate which is often used as a model to study these processes in vitro and in vivo.
Pathways
Ubiquitylation integrates deeply into cell signaling and degradation pathways. This process is key in the ubiquitin-proteasome pathway which is essential for protein breakdown. It is also involved in the Wnt signaling pathway where it affects the turnover of specific proteins like β-catenin. Other proteins such as p53 are regulated by ubiquitylation controlling their stability and activity.
Aberrant ubiquitylation can lead to cancer and neurodegenerative diseases. For instance the dysregulation of p53 ubiquitination contributes to the development of various cancers due to impaired cellular apoptosis. Similarly anomalies in ubiquitylation pathways often relate to Parkinson’s disease where proteins like parkin an E3 ligase are involved. Understanding these connections provides insights into potential therapeutic targets for correcting these dysfunctions.
