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Rac1 G-LISA Activation Assay Kit (Colorimetric Based) 96 assays

Rac1 G-LISA Activation Assay (Colorimetric Based)
$0.00
SKU
BK128
Datasheet
MSDS
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The proprietary Rac1 G-LISA™ Activation Assay that is faster, easier and more precise than traditional pull-down methods to measure Rac1 Activation.

  • Specific for Rac1
  • Colorimetric Based
  • Fast Results
  • Extremely Sensitive

General Information

This Rac1 G-LISA™ is a colorimetric based assay and shares the many advantages that all G-LISA™ activation assay have. This assay is specific for Rac1.

The Rac1 G-LISA™ kit contains a Rac-GTP-binding protein linked to the wells of a 96 well plate. Active GTP-bound Rac1 is captured by this protein while inactive GDP-bound Rac1 is removed following washes. The active Rac1 bound to the wells is detected with a Rac1 specific antibody. The degree of Rac1 activation is determined by comparing readings from activated cell lysates versus non-activated cell lysates.

Click Here for Detailed Methods
BK128prot

Figure 1.The simple procedure of the Rac1 G-LISA™ assay.

Kit Contents - Enough reagents for 96 assays (Stripwell)

  • 96 individual Rac1-GTP binding wells
  • Anti-Rac1 antibody (Cat. # ARC03)
  • Secondary Antibody - HRP
  • Rac1 control protein
  • Cell Lysis Buffer
  • Wash buffer
  • Antigen Presenting Buffer
  • Antibody Dilution Buffer
  • HRP Detection and Stop Reagents
  • Precision Red™ Advanced Protein Assay (Cat. # ADV02)
  • Protease Inhibitor Cocktail (Cat. # PIC02)

 

 

Go to main G-LISA™ page

bk128fig1

Figure 2. Rac1 activation by EGF measured by G-LISA™. Swiss 3T3 cells were serum starved (SS) for 48 h and treated with EGF (10 ng/ml for 2 min). 12.5 μg and 25 μg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm.

For product Datasheets and MSDSs please click on the PDF links below.   

 

    AuthorTitleJournalYearArticle Link
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    Tanaka, Masato et al.Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseasesCommunications Biology 2024
    Wu, Yi et al.Decreased Tiam1-mediated Rac1 activation is responsible for impaired directional persistence of chondrocyte migration in microtiaJournal of Cellular and Molecular Medicine2024
    Szigeti, Kinga et al.CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganizationeBioMedicine2024
    Raykhel, I. et al.HIF2α-dependent Dock4/Rac1-signaling regulates formation of adherens junctions and cell polarity in normoxiaScientific Reports 2024
    Liu, Xiaomin et al.RABV induces biphasic actin cytoskeletal rearrangement through Rac1 activity modulationJournal of Virology2024
    Koenis, Duco Steven et al.Efferocyte-Derived MCTRs Metabolically Prime Macrophages for Continual Efferocytosis via Rac1-Mediated Activation of GlycolysisAdvanced Science2024
    Mendoza-Soto, Pablo et al.Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of PodocytesCells2024
    Vahldieck, Carl et al.Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axisFrontiers in Immunology2024
    Wu, Fei et al.Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflightNature Communications2024
    Lee, Choong Jae et al.The dysadherin/FAK axis promotes individual cell migration in colon cancerInternational Journal of Biological Sciences2024
    Laragione, Teresina et al.KIF1C and new Huntingtin-interacting protein 1 binding proteins regulate rheumatoid arthritis fibroblast-like synoviocytes’ phenotypesFrontiers in Immunology2024
    Moztarzadeh, Sina et al.Cortactin is in a complex with VE-cadherin and is required for endothelial adherens junction stability through Rap1/Rac1 activationScientific Reports2024
    Hong, Geunhye et al.Campylobacter jejuni Modulates Reactive Oxygen Species Production and NADPH Oxidase 1 Expression in Human Intestinal Epithelial CellsCellular Microbiology2023
    Adhicary, Subhodip et al.Rbpj Deficiency Disrupts Vascular Remodeling via Abnormal Apelin and Cdc42 (Cell Division Cycle 42) Activity in Brain Arteriovenous MalformationStroke2023
    Szigeti, Kinga et al.Neuronal actin cytoskeleton gain of function in the human braineBioMedicine2023
    Xie, Wangkai et al.ASAP1 activates the IQGAP1/CDC42 pathway to promote tumor progression and chemotherapy resistance in gastric cancerCell Death & Disease2023
    Takaya, Kento et al.Compound 13 Promotes Epidermal Healing in Mouse Fetuses via Activation of AMPKBiomedicines2023
    Wang, Yanan et al.PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell ComplexNature Communications 2023
    Toffali, Lara et al.An isoform of the giant protein titin is a master regulator of human T lymphocyte traffickingCell reports2023
    Turgu, Busra et al. The HACE1 E3 ligase mediates RAC1 ‐dependent control of mTOR signaling complexes EMBO reports2023
    Bartos, Katalin et al.Renal FGF23 signaling depends on redox protein Memo1 and promotes orthovanadate-sensitive protein phosphotyrosyl phosphatase activityJournal of Cell Communication and Signaling2023
    St. Paul, Amanda et al.FXR1 regulates vascular smooth muscle cell cytoskeleton, VSMC contractility, and blood pressure by multiple mechanismsCell reports2023
    Castillo-Azofeifa, David et al.A DLG1-ARHGAP31-CDC42 axis is essential for the intestinal stem cell response to fluctuating niche Wnt signalingCell stem cell2023
    Dang, Iren et al.Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3Journal of Cell Science2023
    Tokarz, Victoria L. et al.Palmitate-induced insulin resistance causes actin filament stiffness and GLUT4 mis-sorting without altered Akt signallingJournal of Cell Science2023
    Carbonell, Abigail U. et al.Comparing synaptic proteomes across five mouse models for autism reveals converging molecular similarities including deficits in oxidative phosphorylation and Rho GTPase signalingFrontiers in Aging Neuroscience2023
    Crespo, Grace Velez et al.The Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancerMolecular Medicine2023
    Darp, Revati et al.Oncogenic BRAF induces whole-genome doubling through suppression of cytokinesisNature Communications2022
    Harms, Julia et al.Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasionCancer Gene Therapy 2022
    Francis, Caitlin R. et al.Rab35 governs apicobasal polarity through regulation of actin dynamics during sprouting angiogenesisNature Communications 2022
    Kim, Seunghoon et al.Investigating the Anti-Inflammatory Effects of RCI001 for Treating Ocular Surface Diseases: Insight Into the Mechanism of ActionFrontiers in Immunology2022
    Sugisawa, Erika et al.Nociceptor-derived Reg3γ prevents endotoxic death by targeting kynurenine pathway in microgliaCell Reports2022
    Ma, Yuanyuan et al.Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root developmentDevelopment (Cambridge)2021
    Gurusamy, Malarvizhi et al.G-protein-coupled receptor P2Y10 facilitates chemokine-induced CD4 T cell migration through autocrine/paracrine mediatorsNature Communications2021
    Kinsella, Sinéad et al.Attenuation of apoptotic cell detection triggers thymic regeneration after damageCell Reports2021
    Mao, De Yu et al.CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and functionScience Signaling2021
    Wegscheid, Michelle L. et al.Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesisCell Reports2021
    Vallejo, Daniela et al.Wnt5a modulates dendritic spine dynamics through the regulation of Cofilin via small Rho GTPase activity in hippocampal neuronsJournal of Neurochemistry2021
    Krueger, Irena et al.Reelin amplifies glycoprotein VI activation and alphaiib beta3 integrin outside-in signaling via PLC Gamma 2 and Rho GTPasesArteriosclerosis, Thrombosis, and Vascular Biology2020
    Rong, Zhouyi et al.Activation of FAK/Rac1/Cdc42-GTPase signaling ameliorates impaired microglial migration response to Aβ42 in triggering receptor expressed on myeloid cells 2 loss-of-function murine modelsFASEB Journal2020
    Kowluru, Renu A.Retinopathy in a diet-induced type 2 diabetic rat model and role of epigenetic modificationsDiabetes2020
    Andrews, Madeline G. et al.Mtor signaling regulates the morphology and migration of outer radial glia in developing human cortexeLife2020
    Sheftel, Celeste M. et al.Serotonin stimulated parathyroid hormone related protein induction in the mammary epithelia by transglutaminase-dependent serotonylationPLoS ONE2020
    Choraghe, Rohan P. et al.RHOA-mediated mechanical force generation through Dectin-1Journal of cell science2020
    Malek, Natalia et al.Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) technique shows that non-muscle β and γ actin are not equal in relation to human melanoma cells’ motility and focal adhesion formationInternational Journal of Molecular Sciences2020
    Szrejder, Maria et al.Metformin reduces TRPC6 expression through AMPK activation and modulates cytoskeleton dynamics in podocytes under diabetic conditionsBiochimica et Biophysica Acta - Molecular Basis of Disease2020
    Wilson, Kitchener D. et al.Endogenous Retrovirus-Derived lncRNA BANCR Promotes Cardiomyocyte Migration in Humans and Non-human PrimatesDevelopmental Cell2020
    Angé, Marine et al.α1AMP-Activated Protein Kinase Protects against Lipopolysaccharide-Induced Endothelial Barrier Disruption via Junctional Reinforcement and Activation of the p38 MAPK/HSP27 PathwayInternational Journal of Molecular Sciences2020
    Gokoffski, Kimberly K. et al.Physiologic electrical fields direct retinal ganglion cell axon growth in vitroInvestigative Ophthalmology and Visual Science2019
    Wang, Baomei et al.Macrophage β2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced ColitisCell Reports2019
    Haq, Naila et al.Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neuronsPLoS Biology2019
    Zanin, Juan P. et al.The p75NTR influences cerebellar circuit development and adult behavior via regulation of cell cycle duration of granule cell progenitorsJournal of Neuroscience2019
    Stefanelli, Victoria L. et al.Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasionMatrix Biology2019
    Laufer, Julia M. et al.Chemokine Receptor CCR7 Triggers an Endomembrane Signaling Complex for Spatial Rac ActivationCell Reports2019
    Santhana Kumar, Karthiga et al.TGF-β Determines the Pro-migratory Potential of bFGF Signaling in MedulloblastomaCell Reports2018
    Caggia, Silvia et al.Novel role of Giα2 in cell migration: Downstream of PI3-kinase–AKT and Rac1 in prostate cancer cellsJournal of Cellular Physiology2018
    Suraneni, Praveen K. et al.Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activityBlood Advances2018
    Saito-Reis, Chelsea A. et al.The tetraspanin CD82 regulates bone marrow homing and engraftment of hematopoietic stem and progenitor cellsMolecular Biology of the Cell2018
    Rachubik, Patrycja et al.The TRPC6-AMPK pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytesCellular Physiology and Biochemistry2018
    Dyberg, Cecilia et al.Rho-associated kinase is a therapeutic target in neuroblastomaProceedings of the National Academy of Sciences of the United States of America2017
    Chen, Guo Ping et al.Inhibition of farnesyl pyrophosphate synthase attenuates high glucose-induced vascular smooth muscle cells proliferationMolecular Medicine Reports2017
    Gu, Changkyu et al.Dynamin autonomously regulates podocyte focal adhesion maturationJournal of the American Society of Nephrology2017
    Ubba, Vaibhave et al.RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat ModelReproductive Sciences2017
    Kumar, Vijay et al.Integrin beta8 (ITGB8) activates VAV-RAC1 signaling via FAK in the acquisition of endometrial epithelial cell receptivity for blastocyst implantationScientific Reports2017
    Rom, Slava et al.PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrierJournal of Neuroinflammation2016
    Hamers, Anouk A.J. et al.Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and toleranceBMC Genomics2016
    Xiao, Bin et al.Extracellular translationally controlled tumor protein promotes colorectal cancer invasion and metastasis through Cdc42/JNK/ MMP9 signalingOncotarget2016
    Jones, Eleanor L. et al.Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the Tetraspanins CD82 and CD37The Journal of Immunology2016
    Sroka, Jolanta et al.Lamellipodia and membrane blebs drive efficient electrotactic migration of rat walker carcinosarcoma cells WC 256PLoS ONE2016
    Zhao, Chen Ze et al.Inhibition of farnesyl pyrophosphate synthase improves pressure overload induced chronic cardiac remodelingScientific Reports2016
    Sayyad, Wasim A. et al.The role of Rac1 in the growth cone dynamics and force generation of DRG neuronsPLoS ONE2016
    Rom, Slava et al.The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 ltr inhibition and diminution in Rho GTPase activityFrontiers in Microbiology2015
    Rom, Slava et al.Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditionsJournal of Cerebral Blood Flow and Metabolism2015
    Ochoa-Alvarez, Jhon A. et al.Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanismsOncotarget2015
    Guo, Yuna et al.A novel pharmacologic activity of keto***** for therapeutic benefit in ovarian cancer patientsClinical Cancer Research2015
    Breslin, Jerome W. et al.Involvement of local lamellipodia in endothelial barrier functionPLoS ONE2015
    Herberich, Stefanie E. et al.ANKS1B Interacts with the cerebral cavernous malformation protein-1 and controls endothelial permeability but not sprouting angiogenesisPLoS ONE2015
    Mackay, Joanna L. et al.Simultaneous and independent tuning of RhoA and Rac1 activity with orthogonally inducible promotersIntegrative Biology (United Kingdom)2014
    Marinković, Goran et al.6-mercaptopurine reduces macrophage activation and gut epithelium proliferation through inhibition of GTPase Rac1Inflammatory Bowel Diseases2014
    Marinković, Goran et al.Inhibition of GTPase Rac1 in Endothelium by 6-Mercaptopurine Results in Immunosuppression in Nonimmune Cells: New Target for an Old DrugThe Journal of Immunology2014
    Papke, Christina L. et al.Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-βHuman Molecular Genetics2013
    Kalia, Manjula et al.Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic MechanismJournal of Virology2013
    Rom, Slava et al.Selective activation of cannabinoid receptor 2 in leukocytes suppresses their engagement of the brain endothelium and protects the blood-brain barrierAmerican Journal of Pathology2013
    Dubash, Adi D. et al.The GEF Bcr activates RhoA/MAL signaling to promote keratinocyte differentiation via desmoglein-1Journal of Cell Biology2013
    Ramsay, Alan G. et al.Chronic lymphocytic leukemia cells induce defective LFA-1-directed T-cell motility by altering Rho GTPase signaling that is reversible with lenali******Blood2013
    Chen, Bin et al.Alteration of mevalonate pathway related enzyme expressions in pressure overload-induced cardiac hypertrophy and associated heart failure with preserved ejection fractionCellular Physiology and Biochemistry2013
    Yang, Jian et al.Cardiac-specific overexpression of farnesyl pyrophosphate synthase induces cardiac hypertrophy and dysfunction in miceCardiovascular Research2013
    Chen, Guang et al.Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells (Journal of Biological Chemistry (2012) 2Journal of Biological Chemistry2012
    Greco, Carolina M. et al.Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptorCardiovascular Research2012
    Ramsay, Alan G. et al.Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenali******: Establishing a reversible immune evasion mechanism in human cancerBlood2012
    Chen, Si Meng et al.Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTORBiochemical Pharmacology2012
    Antonov, Alexander S. et al.Regulation of endothelial barrier function by TGF-β type I receptor ALK5: potential role of contractile mechanisms and heat shock protein 90Journal of cellular physiology2012
    Oshikawa, Jin et al.Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cellsAmerican Journal of Physiology - Heart and Circulatory Physiology2012
    Montalvo-Ortiz, Brenda L. et al.Characterization of EHop-016, novel small molecule inhibitor of Rac GTPaseThe Journal of biological chemistry2012
    Stefanini, Lucia et al.Rap1-Rac1 circuits potentiate platelet activationArteriosclerosis, thrombosis, and vascular biology2012
    Arpaia, E. et al.The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and ErkOncogene2012
    Rom, Slava et al.Glycogen synthase kinase 3β inhibition prevents monocyte migration across brain endothelial cells via Rac1-GTPase suppression and down-regulation of active integrin conformationAmerican Journal of Pathology2012
    Sánchez, Elena G. et al.African swine fever virus uses macropinocytosis to enter host cellsPLoS Pathogens2012
    Vives, Virginie et al.The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts2011

    Question 1:  Can I detect isoforms other than RhoA, Rac1 or RalA with these G-LISA activation assays?

    Answer 1:  Yes, the RhoA G-LISA (Cat. # BK124), Rac1 G-LISA (Cat. # BK128) and RalA G-LISA (Cat. # BK129) can be used to detect RhoB or RhoC, Rac 2 or Rac3 or RalB, respectively.  The capture proteins that the wells have been coated with bind all of the isoforms of the respective GTPase.  The specificity of signal is conferred by the specificity of the monoclonal primary antibody utilized.  Use of an isoform-specific monoclonal antibody allows detection of other Rho family isoforms.  Please see this citation for an example of this modified procedure (Hall et al., 2008. Type I Collagen Receptor (α2β1) Signaling Promotes Prostate Cancer Invasion through RhoC GTPase. Neoplasia. 10, 797–803). 

    Basically the researcher would test their specific monoclonal antibody in a western blot first to prove specificity to the alternative isoform of interest.  For example, load RhoA and C for negative controls when testing a RhoB monoclonal antibody.  Then the researcher would use 1:50, 1:200 and 1:500 dilutions of their monoclonal antibody on duplicate cell extracts of activated and control state samples. The researcher would then choose the dilution of monoclonal antibody which gave them the highest ratio of activated:control state.

    A simple activated/control state pair of extracts can be made by growing cells to 50% confluence in serum containing media, washing twice with PBS, preparing lysate and aliquoting and freezing  samples in liquid nitrogen.  With one aliquot, defrost and let stand at room temperature for 60 min to degrade the activated signal to a low basal signal, which will be the control state.  The untreated sample (2nd aliquot) will be considered “activated” which most serum grown cells are.

     

    Question 2:  How many cell culture plates can I process at one time during the lysis step?

    Answer 2:  We recommend that from the point at you add lysis buffer to the plate on ice to aliquoting and snap-freezing the lysate samples in liquid nitrogen, no more than 10 min are allowed to elapse.  After 10 min on ice, we find that GTP bound to GTPases (activated GTPases) undergoes rapid hydrolysis.  Rapid processing at 4°C is essential for accurate and reproducible results.  The following guidelines are useful for rapid lysis of cells.

     Washing

    a.  Retrieve culture dish from incubator, immediately aspirate out all of the media and place firmly on ice.

    b.  Immediately rinse cells with an appropriate volume of ice cold PBS (for Cdc42 activation, skip this step and simply aspirate the media) to remove serum proteins.

    c.  Aspirate off all residual PBS buffer. This is essential so that the Lysis Buffer is not diluted. Correct aspiration requires that the culture dish is placed at a steep angle on ice for 1 min to allow excess PBS to collect in the vessel for complete removal.  As noted, the time period between cell lysis and addition of lysates to the wells is critically important. Take the following precautions:

         1.  Work quickly.

         2.  Keeping solutions and lysates embedded in ice so that the temperature is below 4°C. This helps to minimize changes in signal over time.

         3.  We strongly recommend that cell lysates be immediately frozen after harvest and clarification. A sample of at least 20 μl should be kept on ice for protein concentration measurement. The lysates must be snap frozen in liquid nitrogen and stored at -70°C. Lysates should be stored at -70°C for no longer than 30 days.

         4.  Thawing of cell lysates prior to use in the G-LISA assay should be in a room temperature water bath, followed by rapid transfer to ice and immediate use in the assay.

     

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

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