RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays

RhoA Activation Assay Biochem Kit (bead pull-down format) - 20 Assays
$0.00

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.



      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

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      Guo, Shuyu et al.Trio cooperates with Myh9 to regulate neural crest-derived craniofacial developmentTheranostics2021ISSN 1838-7640
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      Zhan, Fangbiao et al.RhoA enhances osteosarcoma resistance to MPPa-PDT via the Hippo/YAP signaling pathwayCell and Bioscience2021ISSN 2045-3701
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      Moodley, Serisha et al.RET isoform-specific interaction with scaffold protein Ezrin promotes cell migration and chemotaxis in lung adenocarcinomaLung cancer (Amsterdam, Netherlands)2020ISSN 1872--8332
      Wang, Xiaobo et al.Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Non-alcoholic SteatohepatitisCell Metabolism2020ISSN 1932-7420
      Hellinger, Johanna W. et al.Identification of drivers of breast cancer invasion by secretome analysis: insight into CTGF signalingScientific Reports2020ISSN 2045-2322
      Kumagai, Shogo et al.An Oncogenic Alteration Creates a Microenvironment that Promotes Tumor Progression by Conferring a Metabolic Advantage to Regulatory T CellsImmunity2020ISSN 1097-4180
      Wang, Qinglian et al.Thymol alleviates AGEs-induced podocyte injury by a pleiotropic effect via NF-κB-mediated by RhoA/ROCK signalling pathwayCell Adhesion and Migration2020ISSN 1933-6926
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      Wang, Ying et al.Murine epsins play an integral role in podocyte functionJournal of the American Society of Nephrology2020ISSN 1533-3450
      Zhou, Yongping et al.Upregulation of ARHGAP30 attenuates pancreatic cancer progression by inactivating the β-catenin pathwayCancer Cell International2020ISSN 1475-2867
      Kawarazaki, Wakako et al.Salt causes aging-associated hypertension via vascular Wnt5a under Klotho deficiencyJournal of Clinical Investigation2020ISSN 1558-8238
      Ramírez-Ramírez, Danelia et al.Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoaJournal of cellular biochemistry2020ISSN 1097--4644
      Wee, Kenneth et al.Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell–matrix adhesionJournal of Cell Science2020ISSN 1477-9137
      Barabutis, Nektarios et al.Protective mechanism of the selective vasopressin V1A receptor agonist selepressin against endothelial barrier dysfunctionJournal of Pharmacology and Experimental Therapeutics2020ISSN 1521-0103
      Jiao, Yi et al.Mechanism of H2S‑mediated ROCK inhibition of total flavones of Rhododendra against myocardial ischemia injuryExperimental and Therapeutic Medicine2019ISSN 1792--0981
      Schipper, Koen et al.Rebalancing of actomyosin contractility enables mammary tumor formation upon loss of E-cadherinNature Communications2019ISSN 2041-1723
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      Akula, Murali K. et al.Protein prenylation restrains innate immunity by inhibiting Rac1 effector interactionsNature Communications2019ISSN 2041-1723
      Yu, Minhao et al.Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathwayEBioMedicine2019ISSN 2352-3964
      Tanga, Naomi et al.The PTN-PTPRZ signal activates the AFAP1L2-dependent PI3K-AKT pathway for oligodendrocyte differentiation: Targeted inactivation of PTPRZ activity in miceGlia2019ISSN 1098-1136
      Cai, Jing et al.A RhoA–YAP–c-Myc signaling axis promotes the development of polycystic kidney diseaseGenes and Development2018ISSN 1549-5477
      Lee, Sung Kyoung et al.Esrp1-Regulated Splicing of Arhgef11 Isoforms Is Required for Epithelial Tight Junction IntegrityCell Reports2018ISSN 2211-1247
      Ma, Teng jiao et al.CLOCK and BMAL1 stabilize and activate RHOA to promote F-actin formation in cancer cellsExperimental and Molecular Medicine2018ISSN 2092-6413
      Brauer, Philip R. et al.Krüppel-like factor 4 mediates cellular migration and invasion by altering RhoA activityCell Communication and Adhesion2018ISSN 1543-5180
      Wang, Qinglian et al.RhoA/Rho-kinase triggers epithelial-mesenchymal transition in mesothelial cells and contributes to the pathogenesis of dialysis-related peritoneal fibrosisOncotarget2018ISSN 1949-2553
      Zhang, Lei et al.Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA ActivationCellular Physiology and Biochemistry2018ISSN 1421-9778
      Xiang, Xiaoyu et al.Vasodilator-stimulated phosphoprotein promotes liver metastasis of gastrointestinal cancer by activating a β1-integrin-FAK-YAP1/TAZ signaling pathwaynpj Precision Oncology2018Article Link
      Zhang, Xin et al.Fasudil increases temozolomide sensitivity and suppresses temozolomide-resistant glioma growth via inhibiting ROCK2/ABCG2Cell Death and Disease2018ISSN 2041-4889
      Lehman, Heather L. et al.NFkB hyperactivation causes invasion of esophageal squamous cell carcinoma with EGFR overexpression and p120-catenin down-regulationOncotarget2018ISSN 1949-2553
      Dong, Bo et al.Functional redundancy of frizzled 3 and frizzled 6 in planar cell polarity control of mouse hair folliclesDevelopment (Cambridge)2018ISSN 1477-9129
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      Haemmerle, Monika et al.Platelets reduce anoikis and promote metastasis by activating YAP1 signalingNature Communications2017ISSN 2041-1723
      Katic, Jelena et al.The cell adhesion molecule CHL1 interacts with patched-1 to regulate apoptosis during postnatal cerebellar developmentJournal of Cell Science2017ISSN 1477-9137
      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 CancersGastroenterology2017ISSN 1528-0012
      Goodman, Linda et al.Integrin α8 and Pcdh15 act as a complex to regulate cilia biogenesis in sensory cellsJournal of Cell Science2017ISSN 1477-9137
      Islam, Salman Ul et al.PRPF overexpression induces drug resistance through actin cytoskeleton rearrangement and epithelial-mesenchymal transitionOncotarget2017ISSN 1949-2553
      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 Physiology2016ISSN 1522-1466
      Gilbert, James et al.The X-linked autism protein KIAA2022/KIDLIA regulates neurite outgrowth via N-cadherin and δ-catenin signalingeNeuro2016ISSN 2373-2822
      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 Vision2015ISSN 1090-0535
      Wang, Shi Jie et al.CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cellsOncotarget2015ISSN 1949-2553
      Parker, William H. et al.Intracellular sscorbate prevents endothelial barrier permeabilization by thrombinJournal of Biological Chemistry2015ISSN 1083-351X
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      Priya, Rashmi et al.Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctionsNature Cell Biology2015ISSN 1476-4679
      Du, Chang Qing et al.Inhibition of farnesyl pyrophosphate synthase attenuates angiotensin II-induced fibrotic responses in vascular smooth muscle cellsInternational Journal of Molecular Medicine2015ISSN 1791-244X
      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 Pharmacology2015ISSN 1476-5381
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      Activation of RhoA in Alcohol-Induced Intestinal Barrier Dysfunction | Request PDFArticle Link

       

      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