DetailsThe 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 proteinProduct Uses IncludeInvestigate 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 Click Here for Detailed Methods Kit contentsThe 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 resultsThere 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 eventsThe 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:  signalseeker@cytoskeleton.comAssociated 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)About Click on the pdf icon below to download the manual CitationsAuthorTitleJournalYearArticle LinkAfrin, Sadia et al.Sim******* modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradationPharmacological Research2021ISSN 1096-1186Tumova, Lucie et al.The ubiquitin-proteasome system does not regulate the degradation of porcine β-microseminoprotein during sperm capacitationInternational Journal of Molecular Sciences2020ISSN 1422-0067Gu, Chunming et al.Identification of berberine as a novel drug for the treatment of multiple myeloma via targeting UHRF1BMC Biology2020ISSN 1741-7007Chu, Yi et al.O-GlcNAcylation of SIX1 enhances its stability and promotes hepatocellular carcinoma proliferationTheranostics2020ISSN 1838-7640Uchiyama, Makoto et al.O2-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel ResponsesCurrent Biology2020ISSN 1879-0445Sen, Arpita et al.The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalizationJournal of Cell Science2020ISSN 1477-9137Park, Jin Suk et al.Mechanical regulation of glycolysis via cytoskeleton architectureNature2020ISSN 1476-4687FaqsVisit our Signal-Seeker™ Tech Tips and FAQs page for technical tips and frequently asked questions regarding this and other Signal-Seeker™ products click here If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com