Ubiquitylation Assay Kit (HeLa lysate-based) (ab139471) facilitates controlled ubiquitin conjugation of substrate proteins (exogenous or endogenous) of interest through the ubiquitin cascade.
Images
Publications
Filter by
- iScience 26:1057452023Centrosomal protein 120 promotes centrosome amplification and gastric cancer progression via USP54-mediated deubiquitination of PLK4.Applications:Unspecified applicationReactive species:Unspecified reactive speciesChenggang Zhang,Xianxiong Ma,Guanxin Wei,Xiuxian Zhu,Peng Hu,Xiang Chen,Dianshi Wang,Yuan Li,Tuo Ruan,Weikang Zhang,Kaixiong Tao,Chuanqing WuPubMed 36590171
- Nature chemical biology 16:1218-12262020Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.Applications:Unspecified applicationReactive species:Unspecified reactive speciesAnn M Cathcart et. al.PubMed 32807965
Key facts
- Sample types
- Purified protein, Cell Lysate
- Assay type
- Enzyme activity
Associated Products
Select an associated product type
1 product for Alternative Product
What's included?
Recommended products
Ubiquitylation Assay Kit (HeLa lysate-based) (ab139471) facilitates controlled ubiquitin conjugation of substrate proteins (exogenous or endogenous) of interest through the ubiquitin cascade.
Key facts
- Sample types
- Purified protein, Cell Lysate
- Assay type
- Enzyme activity
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -80°C
- Appropriate long-term storage conditions
- -80°C
- Storage information
- -80°C
Notes
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.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
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.
- Associated diseases and disorders
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.
Product promise
We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
1 product image
Western blot - Ubiquitylation Assay Kit (HeLa lysate-based) (ab139471)
Western blot of S100 ubiquitin conjugation assays of both endogenous lysate and exogenously added p53 proteins.
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.
Downloads
Product protocols
- Visit the general protocols
- Visit troubleshooting
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com