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Signal-Seeker™ Ubiquitination Detection Kit (10 assay)

Signal-Seeker Ubquitination Detection Kit (10 assays, immunoprecipitation format)
$0.00
SKU
BK161-S
Datasheet
MSDS
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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

Click Here for Detailed Methods

 

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.
CUB02_product_page_figure

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.com


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.# UBA01B-beads)

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

 

Click on the pdf icon below to download the manual

AuthorTitleJournalYearArticle Link
Otani, Takahito et al.The role of adhesion molecules in osteocalcin-induced effects on glucose and lipid metabolism in adipocytesBiochimica et Biophysica Acta (BBA) - Molecular Cell Research2024
Ruggeri, Edoardo et al.Protein kinase CK2α is overexpressed in classical hodgkin lymphoma, regulates key signaling pathways, PD-L1 and may represent a new target for therapyFrontiers in Immunology2024
Shi, Shujie et al.Control of ENaC ubiquitinationRenal Physiology2024
Kotani, Hiroshi et al.Dual inhibition of SUMOylation and MEK conquers MYC-expressing KRAS-mutant cancers by accumulating DNA damageJournal of Biomedical Science2024
Hołda, Mateusz K. et al.Autophagy and ubiquitin-dependent proteolysis processes in left ventricular mass loss in pulmonary arterial hypertensionScientific Reports2024
Liu, Shiwei et al.O-GlcNAcylated RALY Contributes to Hepatocellular Carcinoma Cells Proliferation by Regulating USP22 mRNA Nuclear ExportInternational Journal of Biological Sciences2024
Yu, Hui et al.MELK aggravates lung adenocarcinoma by regulating EZH2 ubiquitination and H3K27me3 histone methylation of LATS2Journal of Cellular and Molecular Medicine2024
Steinbach, Adriana et al.Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophilaMolecular biology of the cell2024
Hotani, Takuma et al.Involvement of Protein Kinase R in Double-Stranded RNA-Induced Proteasomal Degradation of Hypoxia Inducible Factor-1αInflammation2023
Chen, Shanlin et al.Aloe-emodin targets multiple signaling pathways by blocking ubiquitin-mediated degradation of DUSP1 in nasopharyngeal carcinoma cellsPhytotherapy Research2023
Guo, Qianjin et al.TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradationCell reports2023
Huang, Xiaogang et al.Unfolded protein response inhibits KAT2B/MLKL-mediated necroptosis of hepatocytes by promoting BMI1 level to ubiquitinate KAT2BOpen Medicine2023
Tahaei, Ebrahim et al.Pendrin regulation is prioritized by anion in high-potassium dietsAmerican Journal of Physiology - Renal Physiology2023
Wang, Yuan et al.MTSS1 curtails lung adenocarcinoma immune evasion by promoting AIP4-mediated PD-L1 monoubiquitination and lysosomal degradationCell Discovery 2023
Román-Fernández, Alvaro et al.Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic functionScience Advances2023
Yang Z. et. al.Enhancing PD-L1 Degradation by ITCH during MAPK Inhibitor Therapy Suppresses Acquired ResistanceCancer Discov2022
Kholmanskikh, Stanislav et al.Activation of RhoC by regulatory ubiquitination is mediated by LNX1 and suppressed by LIS1Scientific Reports 2022 12:12022
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 Sciences2022
Afrin, Sadia et al.Sim******* modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradationPharmacological Research2021
Tumova, Lucie et al.The ubiquitin-proteasome system does not regulate the degradation of porcine β-microseminoprotein during sperm capacitationInternational Journal of Molecular Sciences2020
Gu, Chunming et al.Identification of berberine as a novel drug for the treatment of multiple myeloma via targeting UHRF1BMC Biology2020
Chu, Yi et al.O-GlcNAcylation of SIX1 enhances its stability and promotes hepatocellular carcinoma proliferationTheranostics2020
Uchiyama, Makoto et al.O2-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel ResponsesCurrent Biology2020
Sen, Arpita et al.The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalizationJournal of Cell Science2020
Park, Jin Suk et al.Mechanical regulation of glycolysis via cytoskeleton architectureNature2020

Visit 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


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