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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.

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Western blot - Ubiquitylation Assay Kit (AB139467), expandable thumbnail

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Key facts

Sample types

Purified protein

Assay type

Direct

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What's included?

20 Test
Components
10X Ubiquitinylation Buffer
1 x 100 µL
20X Mg-ATP Solution
1 x 50 µL
20X Ubiquitin Activating Enzyme Solution (E1)
1 x 50 µL
20X Ubiquitin Solution (Ub)
1 x 50 µL
2X Non-reducing Gel Loading Buffer
1 x 1.25 mL
Ubiquitin Antibody Solution
1 x 25 µL

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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.

Key facts

Sample types

Purified protein

Assay type

Direct

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

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.

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.

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  • Western blot - Ubiquitylation Assay Kit (ab139467), expandable thumbnail

    Western blot - Ubiquitylation Assay Kit (ab139467)

    Example results for Western blotting.

    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.

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Product protocols

For this product, it's our understanding that no specific protocols are required. You can:

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