Ubiquitination Affinity Beads

Ubiquitination Affinity Beads (40 assays)

As part of the Signal-Seeker™ product line, Ubiquitin affinity beads have been optimized in order to detect endogenous levels of mono- and poly-ubiqutinated proteins, which often represent <1% of the target protein. UBA01B-Beads can enrich all ubiquitinated species with a high affinity, including mono-, multi- and polyubiquitin chains (see Application 1 below). The affinity moeities have been cross-linked to beads for low leaching and cleaner detection of the protein of interest. A comprehensive Signal-Seeker™ Ubiquitination Detection Kit is also available (BK161) and is recommended for first time users.

Note: The product #s have been updated from UBA01 and CUB02 to UBA01B and CUB02B beads due to a reformulation of the bead preservation buffer. Reformulation results in an enhanced bead performance post-lyophilization. The affinity protein formulation on the beads has not changed.

Validation Data: Ubiquitination Affinity Beads White Paper

Each lot of affinity-bead is quality controlled  to provide high batch to batch consistency, see COA documents.

Validated Applications

Application 1: Investigate mono- and poly-ubiquitination
The activity of the Rho family of proteins, including Rac1, is known to be regulated, in part, through ubiquitination (also termed ubiquitylation) events that can lead to signaling pathway regulation via degradation and/or localization of the modified protein (Visvikis, O. et al. 2010. Biol. Cell 102: 377-389. Nethe, M., & Hordijik, P.2010. J. Cell Sci. 123: 4011-4018). In many cases, the GTP-bound active form of Rac1 is the preferred substrate for ubiquitination. For example, it has been shown that cells treated with the bacterial toxin CNF1 leads to constitutive activation of Rac1 and subsequent mono- and poly-ubiquitination (Pop, M. et al. 2004. J. Biol.Chem. 279: 35840-35848).
Using ubiquitin affinty beads (Cat. # UBA01B-beads) as part of the Signal Seeker™ Ubiquitination Detection Kit (Cat # BK161) we examined the ubiquitination of endogenous Rac1 in 3T3 cells treated with CNF1 toxin (Cat # CN04) and found that both mono- and polyubiquitination of Rac1 could be detected from 300 μg of 3T3 cell lysate. The kit offers a user friendly tool to examine mono- and poly-ubiquitination for any target protein.

Figure 1: Swiss 3T3 cells were pre-treated with MG-132, then either untreated or treated with CNF1 (CN04) for 3 hours prior to lysis with BlastR buffer. The BK161 kit was utilized to perform the IP on 300 μg of lysate per condition. Lane 1: 3 μg input untreated lysate, Lane 2: Ubiquitin Affinity beads (UBA01) plus untreated lysate, Lane 3: UBA01 plus CN04 treated lysate, Lane 4: ubiquitin control beads (CUB02) plus untreated lysate, Lane 5: CUB02 plus CN04 treated lysate. Samples were analyzed for Rac1 ubiquitination using an anti-Rac1 antibody.

Each package contains enough ubiquitin beads for 40 reactions and sufficient control beads for 5 reactions.


For more information contact:  signalseeker@cytoskeleton.com

Associated Products:

Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)

Signal-Seeker™ SUMOylation 2/3 Detection Kit (Cat. # BK162)

Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)

Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)

Signal-Seeker™: PTMtrue™ Ubiquitin Antibody (Cat.# AUB01)


    AuthorTitleJournalYearArticle Link
    Nguyen, Kha The et al.The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosisNature Cell Biology 2022ISSN 1476--4679
    Dieter, Sebastian M. et al.Degradation of CCNK/CDK12 is a druggable vulnerability of colorectal cancerCell Reports2021ISSN 2211-1247
    Gu, Chunming et al.Identification of berberine as a novel drug for the treatment of multiple myeloma via targeting UHRF1BMC Biology2020ISSN 1741-7007
    Horita, Henrick et al.Utilizing a comprehensive immunoprecipitation enrichment system to identify an endogenous post-translational modification profile for target proteinsJournal of Visualized Experiments2018ISSN 1940-087X
    Horita, Henrick et al.Utilizing optimized tools to investigate PTM crosstalk: Identifying potential PTM crosstalk of acetylated mitochondrial proteinsProteomes2018ISSN 2227-7382
    Horita, Henrick et al.A simple toolset to identify endogenous post-translational modifications for a target protein: A snapshot of the EGFR signaling pathwayBioscience Reports2017ISSN 1573-4935

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