Signal-Seeker™ Ubiquitination Detection Kit (10 assay)

Signal-Seeker Ubquitination Detection Kit (10 assays, immunoprecipitation format)

The Signal-Seeker™ line of produts have been developed to allow simple analysis of key regulatory protein modifications by specialists and non-specialists alike. The comprehensive Signal-Seeker™ kits provide an affinity bead system to isolate and enrich modified proteins from any given cell or tissue lysate. The enriched protein population is then analyzed by standard western blot procedures using a primary antibody to the target protein

Product Uses Include

  • Investigate transient regulatory mechanisms
  • Measure signalling events of multiple pathway member proteins 
  • Discover new modifications of your protein of interest
  • Gain insight into regulatory mechanisms
  • Measure endogenous or transiently expressed protein signalling events

Validation Data: Ubiquitination Detection Kit White Paper


Kit contents

The Ubiquitination kit contains the following components:

Lysis and protein quantitation stepIP and pre-clear stepWash stepElution stepWestern step

 BlastR™ Lysis Buffer 

 BlastR™ Dilution Buffer

 BlastR™ Filters

 Protease Inhibitor Cocktail

 De-Ubiquitinase inhibitor  Cocktail

 Precision Red™ Protein  Assay Reagent

 Ubiquitination Affinity  Beads

 Ubiquitination Control  Beads





 BlastR™ Wash  Buffer






 Spin Columns

 Bead Elution  Buffer





 Chemiluminescent  Reagent A

 Chemiluminescent  Reagent B

 Anti-ubiquitin-HRP  antibody





Example results

There are many applications for these kits (see next section below), here we describe an interesting application for Rac1 protein:

Application 1: Investigate highly transient regulation events

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 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.

Other experiments that could be attempted in this area of research include:
• Pharmacological investigation of ubiquitinases or de-ubiquitinases involved in Rac1 mono- and poly-ubiquitination
• Investigation of the relationship between Rac1 activation and ubiquitination under a variety of different growth factors or drug treatments.
• Examine the interaction of ubiquitinated Rac1 with effectors.
• Examine crosstalk between ubiquitinated Rac1 and other regulatory PTMs of Rac1.

For more information contact:

Associated Products:

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

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

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

Signal-Seeker™ Ubiquitin Affinity Beads (Cat.# UBA01-beads)

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


Click on the pdf icon below to download the manual

    AuthorTitleJournalYearArticle Link
    Kholmanskikh, Stanislav et al.Activation of RhoC by regulatory ubiquitination is mediated by LNX1 and suppressed by LIS1Scientific Reports 2022 12:12022ISSN 2045--2322
    Day-Walsh, Priscilla E. et al.Transcriptional and Post-Translational Regulation of Junctional Adhesion Molecule-B (JAM-B) in Leukocytes under Inflammatory StimuliInternational Journal of Molecular Sciences2022ISSN 1422-0067
    Afrin, Sadia et al.Sim******* modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradationPharmacological Research2021ISSN 1096-1186
    Tumova, Lucie et al.The ubiquitin-proteasome system does not regulate the degradation of porcine β-microseminoprotein during sperm capacitationInternational Journal of Molecular Sciences2020ISSN 1422-0067
    Gu, Chunming et al.Identification of berberine as a novel drug for the treatment of multiple myeloma via targeting UHRF1BMC Biology2020ISSN 1741-7007
    Chu, Yi et al.O-GlcNAcylation of SIX1 enhances its stability and promotes hepatocellular carcinoma proliferationTheranostics2020ISSN 1838-7640
    Uchiyama, Makoto et al.O2-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel ResponsesCurrent Biology2020ISSN 1879-0445
    Sen, Arpita et al.The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalizationJournal of Cell Science2020ISSN 1477-9137
    Park, Jin Suk et al.Mechanical regulation of glycolysis via cytoskeleton architectureNature2020ISSN 1476-4687

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