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RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format)

RhoA Activation Assay Biochem Kit (bead pull-down format) - 20 Assays
$0.00
SKU
BK036-S
Datasheet
MSDS
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Product Uses Include

  • Analysis of in vivo RhoA activation

Introduction
The Rho switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state.  Understanding the mechanisms that regulate activation / inactivation of the GTPases is of obvious biological significance and is a subject of intense investigation.  The fact that many Rho family effector proteins will specifically recognize the GTP bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors RhoA protein activation.  The assay uses the Rho binding domain (RBD) of the Rho effector protein, Rhotekin.  The RBD motif has been shown to bind specifically to the GTP-bound form of RhoA. The fact that the RBD region of Rhotekin has a high affinity for GTP-RhoA and that Rhotekin binding results in a significantly reduced intrinsic and catalytic rate of GTP hydrolysis make it an ideal tool for affinity purification of GTP-RhoA from cell lysates.  The Rhotekin-RBD protein supplied in this kit contains Rhotekin residues 7-89 and is in the form of a GST fusion protein, which allows one to "pull-down" the Rhotekin-RBD/Rho-GTP complex with brightly colored glutathione affinity beads. The assay therefore provides a simple means of quantitating RhoA activation in cells. The amount of activated RhoA is determined by a western blot using a RhoA specific antibody.

      Click Here for Detailed Methods



      Kit contents
      The kit contains sufficient materials for 20 assays, depending on assay setup, and includes reagents for positive and negative controls.  A larger 80 assay version of this kit is available as Cat. # BK036. The following components are included:

      1. GST-tagged Rhotekin-RBD protein on colored agarose beads (Cat. # RT02)
      2. RhoA-specific monoclonal antibody (Cat. # ARH03)
      3. His-tagged RhoA protein (Cat. # RH01)
      4. GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
      5. GDP
      6. Cell lysis Buffer
      7. Wash Buffer
      8. Loading Buffer
      9. STOP Buffer
      10. Protease inhibitor cocktail (Cat. # PIC02)
      11. Manual with detailed protocols and extensive troubleshooting guide
      beads

      Figure 1. The brightly colored glutathione agarose beads in BK036-S makes the kit easy to use.

      Equipment needed

      1. SDS-PAGE minigel system and western blotting transfer apparatus

      Example results
      The RhoA activation assay was tested by loading the RhoA protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded RhoA is very efficiently precipitated while very little GDP-loaded RhoA is precipitated (Fig. 2).

      bk036fig2

      Figure 2. Results from BK036-S RhoA activation assay. Activated Rho was precipitated and detected in a Western blot using kit BK036-S. The first lane shows a 50 ng recombinant His-tagged RhoA standard (Rec. His-RhoA). The following lanes shows the pull-down of inactive, GDP-loaded RhoA (RhoA-GDP PD) or active, GTPγS-loaded RhoA (RhoA-GTP PD) from equal amounts of cell lysates.

      Please check out the new versions of the Rho Activation Assays and associated products:

      G-LISA™ Products:
      Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
      Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
      Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
      RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
      RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)

      Associated Products:
      Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
      Anti-Rac1 monoclonal antibody (Cat.# ARC03)
      Anti-RhoA monoclonal antibody (Cat.# ARH03)

      For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

      AuthorTitleJournalYearArticle Link
      Olivier, Jean Frederic et al.CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitisCommunications Biology2024
      Ye, Junjie et al.RhoA/ROCK-TAZ Axis regulates bone formation within calvarial trans-sutural distraction osteogenesisCellular Signalling2024
      Bai, Xiaoyuan et al.The extracellular cyclophilin A-integrin β2 complex as a therapeutic target of viral pneumoniaMolecular Therapy2024
      Ramos, Raquel et al.New Insights into the Exosome-Induced Migration of Uveal Melanoma Cells and the Pre-Metastatic Niche Formation in the LiverCancers2024
      Ha, Cuong P. et al.Humanin activates integrin αV–TGFβ axis and leads to glioblastoma progressionCell Death & Disease2024
      Jinsheng, Li et al.Micro/nano topological modification of TiO2 nanotubes activates Thy-1 signaling to control osteogenic differentiation of stem cellsSLAS Discovery2024
      Gan, Wan J. et al.A truncation mutant of adenomatous polyposis coli impairs apical cell extrusion through elevated epithelial tissue tensionCytoskeleton2024
      Wei, Zhewei et al.DDR1 Drives Malignant Progression of Gastric Cancer by Suppressing HIF-1α Ubiquitination and DegradationAdvanced Science2024
      Laurent-Issartel, Carine et al.Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5β1 integrinsExperimental Cell Research2024
      Akhter, Md Zahid et al.FAK regulates tension transmission to the nucleus and endothelial transcriptome independent of kinase activityCell Reports2024
      Liu, Yuhan et al.Isoform-independent promotion of contractility and proliferation, and suppression of survival by with no lysine/K kinases in prostate stromal cellsThe FASEB Journal2024
      Chen, Ye et al.Dynamic nanomechanical characterization of cells in exosome therapyMicrosystems & Nanoengineering 2024
      Chen, Xiaoqing et al.ARHGAP6 Suppresses Breast Cancer Tumor Growth by Promoting Ferroptosis via RhoA-ROCK1-p38 MAPK SignalingFrontiers in Bioscience - Landmark2024
      Guo, Xiong et al.Rabenosyn-5 suppresses non-small cell lung cancer metastasis via inhibiting CDC42 activityCancer Gene Therapy 2024
      Gandhi, Nisha et al.In Vitro Cell Culture Model for Osteoclast Activation during Estrogen WithdrawalInternational Journal of Molecular Sciences2024
      Yang, Ruicheng et al.Egr-1 is a key regulator of the blood-brain barrier damage induced by meningitic Escherichia coliCell Communication and Signaling2024
      Veloso, Alexandra et al.The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafishBMC Biology2024
      Yao, Na et al.An emerging role of arecoline on growth performance, intestinal digestion and absorption capacities and intestinal structural integrity of adult grass carp (Ctenopharyngodon idella)Animal Nutrition2023
      Freiberg, Fabian et al.CAR links hypoxia signaling to improved survival after myocardial infarctionExperimental & Molecular Medicine2023
      Aw, Wen Yih et al.Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formationScience Advances2023
      Dong, Yuqing et al.CCL2 promotes lymphatic metastasis via activating RhoA and Rac1 pathway and predict prognosis to some extent in tongue cancerCancer Biology & Therapy2023
      Lin, Xixi et al.Targeting of G-protein coupled receptor 40 alleviates airway hyperresponsiveness through RhoA/ROCK1 signaling pathway in obese asthmatic miceRespiratory Research2023
      Wang, Jirong et al.RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signalingMolecular Medicine2023
      Zhang, Shuang et al.Tensin-3 is involved in osteogenic versus adipogenic fate of human bone marrow stromal cellsCellular and Molecular Life Sciences2023
      Zhang, Tao et al.Extracellular matrix stiffness mediates uterine repair via the Rap1a/ARHGAP35/RhoA/F-actin/YAP axisCell Communication and Signaling2023
      Zhang, Yaping et al.Integrin β3 directly inhibits the Gα13-p115RhoGEF interaction to regulate G protein signaling and platelet exocytosisNature Communications 2023
      Danielson, Laura S. et al.MiR-130b modulates the invasive, migratory, and metastatic behavior of leiomyosarcomaPLOS ONE2023
      Ravn-Boess, Niklas et al.The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerabilityCell reports2023
      Cittadini, Camilla et al.Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot studyJournal of Cellular and Molecular Medicine2023
      Knop, Juna Lisa et al.Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signalingiScience2023
      Mobley, James A. et al.The SARS-CoV-2 spike S1 protein induces global proteomic changes in ATII-like rat L2 cells that are attenuated by hyaluronanAmerican Journal of Physiology - Lung Cellular and Molecular Physiology2023
      Serwe, Guillaume et al.CNK2 promotes cancer cell motility by mediating ARF6 activation downstream of AXL signallingNature Communications2023
      Oh, Mijung et al.High extracellular glucose promotes cell motility by modulating cell deformability and contractility via the cAMP-RhoA-ROCK axis in human breast cancer cellsMolecular Biology of the Cell2023
      Wang, Yueyang et al.Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cellseLife2023
      Spel, Lotte et al.CDC42 regulates PYRIN inflammasome assemblyCell Reports2022
      Zheng, Cankun et al.CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canag******** for Cardiorenal SyndromeFrontiers in Pharmacology2022
      Hamuro, Junji et al.Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium FailureInvestigative Ophthalmology & Visual Science2022
      Rackow, Ashley R. et al.The Novel Small Molecule BTB Inhibits Pro-Fibrotic Fibroblast Behavior though Inhibition of RhoA ActivityInternational Journal of Molecular Sciences2022
      Vittoria, Marc A. et al.Inactivation of the Hippo tumor suppressor pathway promotes melanomaNature Communications2022
      Usuki, Seigo et al.Konjac Ceramide (kCer)-Mediated Signal Transduction of the Sema3A Pathway Promotes HaCaT Keratinocyte DifferentiationBiology2022
      Yokoyama, Yusuke et al.Crumbs3 is expressed in oral squamous cell carcinomas and promotes cell migration and proliferation by affecting RhoA activityOncology Letters2022
      Swiatlowska, Pamela et al.Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switchingScience Advances2022
      Mahly, Adnan et al.Anillin governs mitotic rounding during early epidermal developmentBMC Biology2022
      Shin, Yuna et al.NPFFR2 Contributes to the Malignancy of Hepatocellular Carcinoma Development by Activating RhoA/YAP SignalingCancers2022
      Almarán, Beatriz et al.Rnd3 Is a Crucial Mediator of the Invasive Phenotype of Glioblastoma Cells Downstream of Receptor Tyrosine Kinase SignallingCells2022
      Soda, Tomohiro et al.Loss of KAP3 decreases intercellular adhesion and impairs intracellular transport of laminin in signet ring cell carcinoma of the stomachScientific Reports 2022 12:12022
      Qian, Zhanyang et al.Activation of gluc****-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injuryInternational Journal of Biological Sciences2022
      Loria, Rossella et al.SEMA6A/RhoA/YAP axis mediates tumor-stroma interactions and prevents response to dual BRAF/MEK inhibition in BRAF-mutant melanomaJournal of Experimental and Clinical Cancer Research2022
      Xu, Weiyi et al.Paxillin promotes breast tumor collective cell invasion through maintenance of adherens junction integrityMolecular Biology of the Cell2022
      Kholmanskikh, Stanislav et al.Activation of RhoC by regulatory ubiquitination is mediated by LNX1 and suppressed by LIS1Scientific Reports 2022 12:12022
      Agbaegbu Iweka, Chinyere et al.The lipid phosphatase-like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory moleculesJournal of Neurochemistry2021
      Rolle, Irene Giulia et al.Heart failure impairs the mechanotransduction properties of human cardiac pericytesJournal of Molecular and Cellular Cardiology2021
      Miao, Chunhui et al.An infection-induced RhoB-Beclin 1-Hsp90 complex enhances clearance of uropathogenic Escherichia coliNature Communications2021
      Guo, Shuyu et al.Trio cooperates with Myh9 to regulate neural crest-derived craniofacial developmentTheranostics2021
      Wang, Junyi et al.Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoACell Death and Disease2021
      Zhan, Fangbiao et al.RhoA enhances osteosarcoma resistance to MPPa-PDT via the Hippo/YAP signaling pathwayCell and Bioscience2021
      Ramírez-Ramírez, Danelia et al.Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoaJournal of cellular biochemistry2020
      Guo, Yaxiu et al.Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoCFASEB Journal2020
      Moodley, Serisha et al.RET isoform-specific interaction with scaffold protein Ezrin promotes cell migration and chemotaxis in lung adenocarcinomaLung cancer (Amsterdam, Netherlands)2020
      Wang, Ruixiao et al.Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cellsLife Sciences2020
      Barabutis, Nektarios et al.Protective mechanism of the selective vas******** V1A receptor agonist selepressin against endothelial barrier dysfunctionJournal of Pharmacology and Experimental Therapeutics2020
      Hellinger, Johanna W. et al.Identification of drivers of breast cancer invasion by secretome analysis: insight into CTGF signalingScientific Reports2020
      Wang, Xiaobo et al.Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Non-alcoholic SteatohepatitisCell Metabolism2020
      Grun, Daniel et al.NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survivalMolecular Carcinogenesis2019
      Jiao, Yi et al.Mechanism of H2S‑mediated ROCK inhibition of total flavones of Rhododendra against myocardial ischemia injuryExperimental and Therapeutic Medicine2019
      Ma, Teng jiao et al.CLOCK and BMAL1 stabilize and activate RHOA to promote F-actin formation in cancer cellsExperimental and Molecular Medicine2018
      Zhang, Xin et al.Fasudil increases temoz******* sensitivity and suppresses temoz*******-resistant glioma growth via inhibiting ROCK2/ABCG2Cell Death and Disease2018
      Lehman, Heather L. et al.NFkB hyperactivation causes invasion of esophageal squamous cell carcinoma with EGFR overexpression and p120-catenin down-regulationOncotarget2018
      Goodman, Linda et al.Integrin α8 and Pcdh15 act as a complex to regulate cilia biogenesis in sensory cellsJournal of Cell Science2017
      Islam, Salman Ul et al.PRPF overexpression induces drug resistance through actin cytoskeleton rearrangement and epithelial-mesenchymal transitionOncotarget2017
      Cho, Soo Young et al.Sporadic Early-Onset Diffuse Gastric Cancers Have High Frequency of Somatic CDH1 Alterations, but Low Frequency of Somatic RHOA Mutations Compared With Late-Onset CancersGastroenterology2017
      Watts, Bruns A. et al.High-mobility group box 1 inhibits HCO3_ absorption in the medullary thick ascending limb through RAGE-rho-ROCK-mediated inhibition of basolateral Na+/H+ exchangeAmerican Journal of Physiology - Renal Physiology2016
      Gilbert, James et al.The X-linked autism protein KIAA2022/KIDLIA regulates neurite outgrowth via N-cadherin and δ-catenin signalingeNeuro2016
      Wang, Y. et al.Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac, NSC23766 and EHT1864British Journal of Pharmacology2015
      Su, Chien Chia et al.Phenotypes of trypsin- and collagenase-prepared bovine corneal endothelial cells in the presence of a selective rho kinase inhibitor, Y-27632Molecular Vision2015
      Wang, Shi Jie et al.CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cellsOncotarget2015
      Yan, Chao et al.Discovery and characterization of small molecules that target the GTPase RalNature2014
      Stankiewicz, Trisha R. et al.Rho family GTPases: Key players in neuronal development, neuronal survival, and neurodegenerationFrontiers in Cellular Neuroscience2014
      Tong, Jing et al.Activation of RhoA in Alcohol-Induced Intestinal Barrier DysfunctionInflammation2013
      Zhan, Hong et al.The effect of an NgR1 antagonist on the neuroprotection of cortical axons after cortical infarction in ratsNeurochemical Research2013
      Xie, Xi et al.Activation of RhoA/ROCK regulates NF-κB signaling pathway in experimental diabetic nephropathyMolecular and cellular endocrinology2013
      Hara, Yusuke et al.Directional migration of leading-edge mesoderm generates physical forces: Implication in Xenopus notochord formation during gastrulationDevelopmental Biology2013
      Nithipatikom, Kasem et al.Cannabinoid receptor type 1 (CB1) activation inhibits small GTPase RhoA activity and regulates motility of prostate carcinoma cellsEndocrinology2012
      Lee, Wonhwa et al.Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal modelsToxicology and applied pharmacology2012
      Li, B. et al.RhoA triggers a specific signaling pathway that generates transforming microvesicles in cancer cellsOncogene2012
      Cheng, Haoran et al.Infrasonic noise induces axonal degeneration of cultured neurons via a Ca²⁺ influx pathwayToxicology letters2012
      Gastonguay, Adam et al.The role of Rac1 in the regulation of NF-kB activity, cell proliferation, and cell migration in non-small cell lung carcinomaCancer Biology & Therapy2012
      Tsuboi, Naoko et al.The effect of monocyte chemoattractant protein-1/CC chemokine ligand 2 on aqueous humor outflow facilityInvestigative Ophthalmology and Visual Science2012
      Khan, Omar M. et al.Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in miceJournal of Clinical Investigation2011
      Hu, Yi et al.Effects of ischemic preconditioning on vascular reactivity and calcium sensitivity after hemorrhagic shock and their relationship to the RhoA–Rho-kinase pathway in ratsJournal of cardiovascular pharmacology2011
      Balanis, Nikolas et al.β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cellsMolecular Biology of the Cell2011
      Ríos, Amelia et al.Participation of Rho, ROCK-2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signalingCell Biology International2008
      Cheng, Zhi Jie et al.Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirementsMolecular Biology of the Cell2006
      Pixley, Fiona J. et al.BCL6 suppresses RhoA activity to alter macrophage morphology and motilityJournal of cell science2005
      Sasai, Noriaki et al.The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movementsNature Cell Biol2004
      Yang, Seun Ah et al.Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in plateletsJournal of Biological Chemistry2004
      Orr, Anthony Wayne et al.Thrombospondin Induces RhoA Inactivation through FAK-dependent Signaling to Stimulate Focal Adhesion DisassemblyJournal of Biological Chemistry2004
      Cetin, Selma et al.Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesionsThe Journal of biological chemistry2004
      Birukova, Anna A. et al.Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassemblyAmerican journal of physiology. Lung cellular and molecular physiology2004
      Setiadi, Hendra et al.Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flowJournal of Cell Biology2003

       

      Question 1: I have high background and/or multiple bands on my western blot.  How can I fix this?

      Answer 1:  There are multiple causes of high background and/or multiple bands.  Some suggestions to improve background signal include:

      1.   When blotting use 70v for 45min only as the small G-proteins are very mobile.
      2. Fully remove SDS from the gel by using a non-SDS containing buffer for transfer and performing a full 15 min gel wash step in the transfer buffer before blotting.
      3. Dry the PVDF membrane for 30 min after transfer and before blocking (not necessary for nitrocellulose)
      4. Making sure that the TBST contains 10 mM Tris, 0.05% Tween 20 and 150 mM NaCl.
      5. Incubating with the primary antibody overnight at 4°C and using the appropriate ECL detection system. 

       

      Question 2: How much of the beads should I use for my pull-down experiments?

      Answer 2:  Rhotekin-RBD beads (Cat. # RT02) will bind to Rho-GDP with a much lower affinity than Rho-GTP.  If too many rhotekin-RBD beads are added to the pull-down assay there will be significant binding to inactive (GDP-bound) RhoA.  The result of this will be an underestimation of RhoA activation.  For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given RhoA activation or inactivation assay.  Once optimal conditions have been established, bead titrations should no longer be necessary. We recommend 25, 50 and 100 μg bead titrations.

       

      Question 3:  How can I test whether the beads are working properly?

      Answer 3:  A standard biological assay for rhotekin-RBD beads consists of a RhoA protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP.  Here are guidelines to follow (see Cat. # RT02 and BK036 datasheets for more details):

       

      Positive Cellular Protein Control:

      Total cell lysate (300 – 800 μg) should be loaded with GTPγS as a positive control for the pull-down assay.  The following reaction details how to load endogenous RhoA with the nonhydrolysable GTP analog (GTPγS).  This is an excellent substrate for rhotekin-RBD beads and should result in a strong positive signal in a pull-down assay.

       

      a) Perform GTP loading on 300 – 800 μg of cell lysate (0.5 mg/ml protein concentration) by adding 1/10th volume of Loading Buffer.

      b) Immediately add 1/100th volume of GTPγS (200 μM final concentration).  Under these conditions, 5 - 10% of the RhoA protein will load with non-hydrolysable GTPγS and will be “pulled-down” with the rhotekin-RBD beads in the assay.

      c) Incubate the control sample at 30°C for 15 min with gentle rotation.

      d) Stop the reaction by transferring the tube to 4°C and adding 1/10th volume of STOP Buffer.

      e) Use this sample immediately in a pull-down assay.

       

      Negative Cellular Protein Control:

      This reaction should be performed in an identical manner to the Positive Control reaction except that 1/100th volume of GDP (1 mM final concentration) should be added to the reaction in place of the GTPγS.  Loading endogenous RhoA with GDP will inactivate RhoA and this complex will bind very poorly to rhotekin-RBD beads.

       

       

      If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com

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