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RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) - 80 Assays

RhoA Activation Assay Biochem Kit (bead pull-down format) - 80 Assays
$0.00
SKU
BK036
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 80 assays depending on activation levels of Rho in cells and includes reagents for positive and negative controls. 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 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).

bk034fig2

Figure 2. Results from BK036 RhoA activation assay. Activated Rho was precipitated and detected in a Western blot using kit BK036. 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 version of the Rac Activation Assay 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
Spel, Lotte et al.CDC42 regulates PYRIN inflammasome assemblyCell Reports2022ISSN 2211--1247
Almarán, Beatriz et al.Rnd3 Is a Crucial Mediator of the Invasive Phenotype of Glioblastoma Cells Downstream of Receptor Tyrosine Kinase SignallingCells2022ISSN 2073-4409
Zheng, Cankun et al.CX3CL1 Worsens Cardiorenal Dysfunction and Serves as a Therapeutic Target of Canagliflozin for Cardiorenal SyndromeFrontiers in Pharmacology2022ISSN 1663-9812
Soda, Tomohiro et al.Loss of KAP3 decreases intercellular adhesion and impairs intracellular transport of laminin in signet ring cell carcinoma of the stomachScientific Reports2022ISSN 2045-2322
Kholmanskikh, Stanislav et al.Activation of RhoC by regulatory ubiquitination is mediated by LNX1 and suppressed by LIS1Scientific Reports 2022 12:12022ISSN 2045--2322
Xu, Weiyi et al.Paxillin promotes breast tumor collective cell invasion through maintenance of adherens junction integrityMolecular Biology of the Cell2022ISSN 1939-4586
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 Research2022ISSN 1756-9966
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 12:12022ISSN 2045--2322
Qian, Zhanyang et al.Activation of glucagon-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 Sciences2022ISSN 1449-2288
Shin, Yuna et al.NPFFR2 Contributes to the Malignancy of Hepatocellular Carcinoma Development by Activating RhoA/YAP SignalingCancers2022ISSN 2072-6694
Mahly, Adnan et al.Anillin governs mitotic rounding during early epidermal developmentBMC Biology2022ISSN 1741-7007
Swiatlowska, Pamela et al.Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switchingScience Advances2022ISSN 2375-2548
Yokoyama, Yusuke et al.Crumbs3 is expressed in oral squamous cell carcinomas and promotes cell migration and proliferation by affecting RhoA activityOncology Letters2022ISSN 1792-1082
Usuki, Seigo et al.Konjac Ceramide (kCer)-Mediated Signal Transduction of the Sema3A Pathway Promotes HaCaT Keratinocyte DifferentiationBiology2022ISSN 2079-7737
Vittoria, Marc A. et al.Inactivation of the Hippo tumor suppressor pathway promotes melanomaNature Communications 2022 13:12022ISSN 2041--1723
Rackow, Ashley R. et al.The Novel Small Molecule BTB Inhibits Pro-Fibrotic Fibroblast Behavior though Inhibition of RhoA ActivityInternational Journal of Molecular Sciences2022ISSN 1422-0067
Hamuro, Junji et al.Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium FailureInvestigative Ophthalmology & Visual Science2022ISSN 1552--5783
Seetharaman, Shailaja et al.Microtubules tune mechanosensitive cell responsesNature Materials2022ISSN 1476-4660
Lee, Jiyoung et al.Role of MCP-1 and IL-8 in viral anterior uveitis, and contractility and fibrogenic activity of trabecular meshwork cellsScientific Reports2021ISSN 2045-2322
Miao, Chunhui et al.An infection-induced RhoB-Beclin 1-Hsp90 complex enhances clearance of uropathogenic Escherichia coliNature Communications2021ISSN 2041-1723
Guo, Shuyu et al.Trio cooperates with Myh9 to regulate neural crest-derived craniofacial developmentTheranostics2021ISSN 1838-7640
Wang, Junyi et al.Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoACell Death and Disease2021ISSN 2041-4889
Zhan, Fangbiao et al.RhoA enhances osteosarcoma resistance to MPPa-PDT via the Hippo/YAP signaling pathwayCell and Bioscience2021ISSN 2045-3701
Chen, Lei et al.The adenosine A2A receptor alleviates postoperative delirium-like behaviors by restoring blood cerebrospinal barrier permeability in ratsJournal of Neurochemistry2021ISSN 1471-4159
Ko, Eunsun et al.Nrf2 regulates cell motility through RhoA–ROCK1 signalling in non-small-cell lung cancer cellsScientific Reports2021ISSN 2045-2322
Nishiyama, Kanako et al.CNKSR1 serves as a scaffold to activate an EGFR phosphatase via exclusive interaction with RhoB-GTPLife Science Alliance2021ISSN 2575-1077
Sharma, Pawan et al.Diacylglycerol kinase inhibition reduces airway contraction by negative feedback regulation of gq-signalingAmerican Journal of Respiratory Cell and Molecular Biology2021ISSN 1535-4989
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 Neurochemistry2021ISSN 1471-4159
De Belly, Henry et al.Membrane Tension Gates ERK-Mediated Regulation of Pluripotent Cell FateCell Stem Cell2021ISSN 1875-9777
Rolle, Irene Giulia et al.Heart failure impairs the mechanotransduction properties of human cardiac pericytesJournal of Molecular and Cellular Cardiology2021ISSN 0022--2828
Wang, Ying et al.Murine epsins play an integral role in podocyte functionJournal of the American Society of Nephrology2020ISSN 1533-3450
Wee, Kenneth et al.Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell–matrix adhesionJournal of Cell Science2020ISSN 1477-9137
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
Kitchen, Gareth B. et al.The clock gene Bmal1 inhibits macrophage motility, phagocytosis, and impairs defense against pneumoniaProceedings of the National Academy of Sciences of the United States of America2020ISSN 1091-6490
Wang, Ruixiao et al.Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cellsLife Sciences2020ISSN 1879-0631
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
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
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
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
Guo, Yaxiu et al.Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoCFASEB Journal2020ISSN 1530-6860
Ramírez-Ramírez, Danelia et al.Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoaJournal of cellular biochemistry2020ISSN 1097--4644
Grun, Daniel et al.NRP-1 interacts with GIPC1 and SYX to activate p38 MAPK signaling and cancer stem cell survivalMolecular Carcinogenesis2019ISSN 1098-2744
Akula, Murali K. et al.Protein prenylation restrains innate immunity by inhibiting Rac1 effector interactionsNature Communications2019ISSN 2041-1723
Schipper, Koen et al.Rebalancing of actomyosin contractility enables mammary tumor formation upon loss of E-cadherinNature Communications2019ISSN 2041-1723
Jiao, Yi et al.Mechanism of H2S‑mediated ROCK inhibition of total flavones of Rhododendra against myocardial ischemia injuryExperimental and Therapeutic Medicine2019ISSN 1792--0981
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
Yu, Minhao et al.Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathwayEBioMedicine2019ISSN 2352-3964
Lee, Sung Kyoung et al.Esrp1-Regulated Splicing of Arhgef11 Isoforms Is Required for Epithelial Tight Junction IntegrityCell Reports2018ISSN 2211-1247
Cai, Jing et al.A RhoA–YAP–c-Myc signaling axis promotes the development of polycystic kidney diseaseGenes and Development2018ISSN 1549-5477
Zhang, Xin et al.Fasudil increases temozolomide sensitivity and suppresses temozolomide-resistant glioma growth via inhibiting ROCK2/ABCG2Cell Death and Disease2018ISSN 2041-4889
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
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
Lehman, Heather L. et al.NFkB hyperactivation causes invasion of esophageal squamous cell carcinoma with EGFR overexpression and p120-catenin down-regulationOncotarget2018ISSN 1949-2553
Haemmerle, Monika et al.Platelets reduce anoikis and promote metastasis by activating YAP1 signalingNature Communications2017ISSN 2041-1723
Goodman, Linda et al.Integrin α8 and Pcdh15 act as a complex to regulate cilia biogenesis in sensory cellsJournal of Cell Science2017ISSN 1477-9137
Xue, Li et al.Duration of simulated microgravity affects the differentiation of mesenchymal stem cellsMolecular Medicine Reports2017ISSN 1791-3004
Islam, Salman Ul et al.PRPF overexpression induces drug resistance through actin cytoskeleton rearrangement and epithelial-mesenchymal transitionOncotarget2017ISSN 1949-2553
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
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
Priya, Rashmi et al.Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctionsNature Cell Biology2015ISSN 1476-4679
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
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
Parker, William H. et al.Intracellular sscorbate prevents endothelial barrier permeabilization by thrombinJournal of Biological Chemistry2015ISSN 1083-351X
Inoue-Mochita, Miyuki et al.P38 MAP kinase inhibitor suppresses transforming growth factor-β2-induced type 1 collagen production in trabecular meshwork cellsPLoS ONE2015ISSN 1932-6203
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
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
Stankiewicz, Trisha R. et al.Rho family GTPases: Key players in neuronal development, neuronal survival, and neurodegenerationFrontiers in Cellular Neuroscience2014ISSN 1662-5102
Yan, Chao et al.Discovery and characterization of small molecules that target the GTPase Ral2014ISSN 1476--4687
Xie, Xi et al.Activation of RhoA/ROCK regulates NF-κB signaling pathway in experimental diabetic nephropathyMolecular and cellular endocrinology2013ISSN 1872--8057
Zhan, Hong et al.The effect of an NgR1 antagonist on the neuroprotection of cortical axons after cortical infarction in ratsNeurochemical Research2013ISSN 0364-3190
Hara, Yusuke et al.Directional migration of leading-edge mesoderm generates physical forces: Implication in Xenopus notochord formation during gastrulationDevelopmental Biology2013ISSN 1095-564X
Xu, Jie et al.RhoGAPs Attenuate Cell Proliferation by Direct Interaction with p53 Tetramerization DomainCell Reports2013ISSN 2211-1247
Tsuboi, Naoko et al.The effect of monocyte chemoattractant protein-1/CC chemokine ligand 2 on aqueous humor outflow facilityInvestigative Ophthalmology and Visual Science2012ISSN 0146-0404
Nithipatikom, Kasem et al.Cannabinoid receptor type 1 (CB1) activation inhibits small GTPase RhoA activity and regulates motility of prostate carcinoma cellsEndocrinology2012ISSN 1945--7170
Lee, Wonhwa et al.Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal modelsToxicology and applied pharmacology2012ISSN 1096--0333
Dong, Ming et al.Increased Rho kinase activity in congestive heart failureEuropean Journal of Heart Failure2012ISSN 1388-9842
Cheng, Haoran et al.Infrasonic noise induces axonal degeneration of cultured neurons via a Ca²⁺ influx pathwayToxicology letters2012ISSN 1879--3169
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 & Therapy2012ISSN 1538--4047
Li, B. et al.RhoA triggers a specific signaling pathway that generates transforming microvesicles in cancer cellsOncogene2012ISSN 1476--5594
Eitaki, Masato et al.Vincristine enhances amoeboid-like motility via GEF-H1/RhoA/ROCK/Myosin light chain signaling in MKN45 cellsBMC Cancer2012ISSN 1471-2407
Borah, Sapan et al.The Rho1 GTPase-activating protein CgBem2 is required for survival of azole stress in Candida glabrataJournal of Biological Chemistry2011ISSN 0021-9258
Khan, Omar M. et al.Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in miceJournal of Clinical Investigation2011ISSN 0021-9738
Balanis, Nikolas et al.β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cellsMolecular Biology of the Cell2011ISSN 1059-1524
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 pharmacology2011ISSN 1533--4023
Du, Hui et al.Phenylephrine induces elevated RhoA activation and smooth muscle α-actin expression in Pkd2+/-vascular smooth muscle cellsHypertension Research2010ISSN 0916-9636
Xing, Dongmei et al.Effect of cAMP on TGFβ1-induced corneal keratocyte-myofibroblast transformationInvestigative Ophthalmology and Visual Science2009ISSN 0146-0404
Xing, Dongmei et al.Hypoxia reduces TGFβ1-induced corneal keratocyte myofibroblast transformationMolecular Vision2009ISSN 1090-0535
Ríos, Amelia et al.Participation of Rho, ROCK-2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signalingCell Biology International2008ISSN 1065-6995
Cheng, Zhi Jie et al.Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirementsMolecular Biology of the Cell2006ISSN 1059-1524
Pixley, Fiona J. et al.BCL6 suppresses RhoA activity to alter macrophage morphology and motilityJournal of cell science2005ISSN 0021--9533
Sasai, Noriaki et al.The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements2004PMID 15258592
Birukova, Anna A. et al.Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassemblyAmerican journal of physiology. Lung cellular and molecular physiology2004ISSN 1040--0605
Cetin, Selma et al.Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesionsThe Journal of biological chemistry2004ISSN 0021--9258
Yang, Seun Ah et al.Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in plateletsJournal of Biological Chemistry2004ISSN 0021-9258
Orr, Anthony Wayne et al.Thrombospondin Induces RhoA Inactivation through FAK-dependent Signaling to Stimulate Focal Adhesion DisassemblyJournal of Biological Chemistry2004ISSN 0021--9258
Setiadi, Hendra et al.Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flowJournal of Cell Biology2003ISSN 0021--9525
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

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