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    ubiquitylation-assay-kit-ab139467.pdf

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Ubiquitylation Assay Kit (ab139467)

  • Datasheet
  • SDS
  • Protocol Booklet
Submit a review Q&A (1)References (2)

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Example results for Western blotting

    Key features and details

    • Assay type: Direct
    • Sample type: Purified protein

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    Overview

    • Product name

      Ubiquitylation Assay Kit
      See all Ubiquitylation kits
    • Sample type

      Purified protein
    • Assay type

      Direct
    • Product overview

      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

    • 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.

    Properties

    • Storage instructions

      Please refer to protocols.
    • Components 20 tests
      10X Ubiquitinylation Buffer 1 x 100µl
      20X Mg-ATP Solution 1 x 50µl
      20X Ubiquitin Activating Enzyme Solution (E1) 1 x 50µl
      20X Ubiquitin Solution (Ub) 1 x 50µl
      2X Non-reducing Gel Loading Buffer 1 x 1.25ml
      Ubiquitin Antibody Solution 1 x 25µl
    • Research areas

      • Kits/ Lysates/ Other
      • Kits
      • Epigenetic kits
      • Ubiquitin kits

    Associated products

    • Related Products

      • Goat Anti-Mouse IgG H&L (HRP) (ab6789)

    Images

    • Example results for Western blotting
      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.

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
    • SDS
  • References (2)

    Publishing research using ab139467? Please let us know so that we can cite the reference in this datasheet.

    ab139467 has been referenced in 2 publications.

    • Arora H  et al. The ATP-Binding Cassette Gene ABCF1 Functions as an E2 Ubiquitin-Conjugating Enzyme Controlling Macrophage Polarization to Dampen Lethal Septic Shock. Immunity 50:418-431.e6 (2019). PubMed: 30770245
    • Zuo Q  et al. Interaction of the primordial germ cell-specific protein C2EIP with PTCH2 directs differentiation of embryonic stem cells via HH signaling activation. Cell Death Dis 9:497 (2018). PubMed: 29703892

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    Question

    Does the 20X Ubiquitin Solution (Ub) contains unlinked ubiquitin or is it linked? And if its linked, can you please provide further details on the linkage.

    Read More

    Abcam community

    Verified customer

    Asked on Oct 06 2014

    Answer

    The 20X Ub solution is unlinked monomer. The E1 enzyme activation is a thioester link with mono-Ub, which is transferd to an E2 enzyme. E1 itself is not ubiquitinated, but the thioester link remains attached until the transfer happens to E2 after ATP is introduced.

    Read More

    Jeremy Kasanov

    Abcam Scientific Support

    Answered on Oct 06 2014

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