Rac1 G-LISA Activation Assay Kit (Colorimetric Based) 24 assays

Rac1 G-LISA Activation Assay (Colorimetric Based)
$0.00

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.

Kit Contents - Enough reagents for 24 assays (Stripwell)

  • 24 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)
BK128prot

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

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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    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 Reports2021ISSN 2211-1247
    Mao, De Yu et al.CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and functionScience Signaling2021ISSN 1937-9145
    Kinsella, Sinéad et al.Attenuation of apoptotic cell detection triggers thymic regeneration after damageCell Reports2021ISSN 2211-1247
    Ma, Shang et al.A role of PIEZO1 in iron metabolism in mice and humansCell2021ISSN 1097-4172
    Gurusamy, Malarvizhi et al.G-protein-coupled receptor P2Y10 facilitates chemokine-induced CD4 T cell migration through autocrine/paracrine mediatorsNature Communications2021ISSN 2041-1723
    Vallejo, Daniela et al.Wnt5a modulates dendritic spine dynamics through the regulation of Cofilin via small Rho GTPase activity in hippocampal neuronsJournal of Neurochemistry2021ISSN 1471-4159
    Merlin, Johanna et al.Non-canonical glutamine transamination sustains efferocytosis by coupling redox buffering to oxidative phosphorylationNature Metabolism2021ISSN 2522-5812
    Ma, Yuanyuan et al.Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root developmentDevelopment (Cambridge)2021ISSN 1477-9129
    Choraghe, Rohan P. et al.RHOA-mediated mechanical force generation through Dectin-1Journal of cell science2020ISSN 1477-9137
    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 Disease2020ISSN 1879-260X
    Sheftel, Celeste M. et al.Serotonin stimulated parathyroid hormone related protein induction in the mammary epithelia by transglutaminase-dependent serotonylationPLoS ONE2020ISSN 1932-6203
    Andrews, Madeline G. et al.Mtor signaling regulates the morphology and migration of outer radial glia in developing human cortexeLife2020ISSN 2050-084X
    Wilson, Kitchener D. et al.Endogenous Retrovirus-Derived lncRNA BANCR Promotes Cardiomyocyte Migration in Humans and Non-human PrimatesDevelopmental Cell2020ISSN 1878-1551
    Kowluru, Renu A.Retinopathy in a diet-induced type 2 diabetic rat model and role of epigenetic modificationsDiabetes2020ISSN 1939-327X
    Larribère, Lionel et al.NF1-RAC1 axis regulates migration of the melanocytic lineageTranslational Oncology2020ISSN 1936-5233
    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 Journal2020ISSN 1530-6860
    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 Sciences2020ISSN 1422-0067
    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 Sciences2020ISSN 1422-0067
    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 Biology2020ISSN 1524-4636
    Woida, Patrick J. et al.The Vibrio cholerae MARTX toxin silences the inflammatory response to cytoskeletal damage before inducing actin cytoskeleton collapseScience Signaling2020ISSN 1937-9145
    Chen, Guo Ping et al.Geranylgeranyl Transferase-I Knockout Inhibits Oxidative Injury of Vascular Smooth Muscle Cells and Attenuates Diabetes-Accelerated AtherosclerosisJournal of Diabetes Research2020ISSN 2314-6753
    Baker, Martin J. et al.P-REX1-independent, calcium-dependent RAC1 hyperactivation in prostate cancerCancers2020ISSN 2072-6694
    Stefanelli, Victoria L. et al.Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasionMatrix Biology2019ISSN 1569-1802
    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 Neuroscience2019ISSN 1529-2401
    Haq, Naila et al.Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neuronsPLoS Biology2019ISSN 1545-7885
    Wu, Wenjuan et al.Inhibition of Rac1-dependent forgetting alleviates memory deficits in animal models of Alzheimer’s diseaseProtein and Cell2019ISSN 1674-8018
    Gokoffski, Kimberly K. et al.Physiologic electrical fields direct retinal ganglion cell axon growth in vitroInvestigative Ophthalmology and Visual Science2019ISSN 1552-5783
    Soni, Upendra Kumar et al.A high level of TGF-B1 promotes endometriosis development via cell migration, adhesiveness, colonization, and invasivenessBiology of Reproduction2019ISSN 1529-7268
    Wang, Baomei et al.Macrophage β2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced ColitisCell Reports2019ISSN 2211-1247
    De Bessa, Tiphany Coralie et al.Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRasCell Death and Disease2019ISSN 2041-4889
    Laufer, Julia M. et al.Chemokine Receptor CCR7 Triggers an Endomembrane Signaling Complex for Spatial Rac ActivationCell Reports2019ISSN 2211-1247
    Shi, Wei et al.EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cellsJournal of Biological Chemistry2019ISSN 1083-351X
    Saito-Reis, Chelsea A. et al.The tetraspanin CD82 regulates bone marrow homing and engraftment of hematopoietic stem and progenitor cellsMolecular Biology of the Cell2018ISSN 1939-4586
    Duraisamy, Arul J. et al.Epigenetics and regulation of oxidative stress in diabetic retinopathyInvestigative Ophthalmology and Visual Science2018ISSN 1552-5783
    Stierwalt, Harrison D. et al.Insulin-stimulated Rac1-GTP binding is not impaired by palmitate treatment in L6 myotubesPhysiological Reports2018ISSN 2051-817X
    Proto, Jonathan D. et al.Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation ResolutionImmunity2018ISSN 1097-4180
    Rachubik, Patrycja et al.The TRPC6-AMPK pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytesCellular Physiology and Biochemistry2018ISSN 1421-9778
    Olabi, Safiah et al.Integrin-Rac signalling for mammary epithelial stem cell self-renewal 06 Biological Sciences 0601 Biochemistry and Cell BiologyBreast Cancer Research2018ISSN 1465-542X
    Mayer, Louisa et al.Nbeal2 interacts with Dock7, Sec16a, and Vac14Blood2018ISSN 1528-0020
    Suraneni, Praveen K. et al.Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activityBlood Advances2018ISSN 2473-9537
    Peretti, Amanda S. et al.The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) MiceAmerican Journal of Pathology2018ISSN 1525-2191
    Santhana Kumar, Karthiga et al.TGF-β Determines the Pro-migratory Potential of bFGF Signaling in MedulloblastomaCell Reports2018ISSN 2211-1247
    Caggia, Silvia et al.Novel role of Giα2 in cell migration: Downstream of PI3-kinase–AKT and Rac1 in prostate cancer cellsJournal of Cellular Physiology2018ISSN 1097-4652
    Dyberg, Cecilia et al.Rho-associated kinase is a therapeutic target in neuroblastomaProceedings of the National Academy of Sciences of the United States of America2017ISSN 1091-6490
    Hendricks, Louie et al.Milk fat globule-epidermal growth factor-factor VIII–derived peptide MSP68 is a cytoskeletal immunomodulator of neutrophils that inhibits Rac1Journal of Surgical Research2017ISSN 1095-8673
    Moreno-Layseca, Paulina et al.The requirement of integrins for breast epithelial proliferationEuropean Journal of Cell Biology2017ISSN 1618-1298
    Kumar, Vijay et al.Integrin beta8 (ITGB8) activates VAV-RAC1 signaling via FAK in the acquisition of endometrial epithelial cell receptivity for blastocyst implantationScientific Reports2017ISSN 2045-2322
    Ubba, Vaibhave et al.RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat ModelReproductive Sciences2017ISSN 1933-7205
    Gu, Changkyu et al.Dynamin autonomously regulates podocyte focal adhesion maturationJournal of the American Society of Nephrology2017ISSN 1533-3450
    Chen, Guo Ping et al.Inhibition of farnesyl pyrophosphate synthase attenuates high glucose-induced vascular smooth muscle cells proliferationMolecular Medicine Reports2017ISSN 1791-3004
    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 Immunology2016ISSN 0022--1767
    Baranov, Maksim V. et al.SWAP70 Organizes the Actin Cytoskeleton and Is Essential for PhagocytosisCell Reports2016ISSN 2211-1247
    Rom, Slava et al.PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrierJournal of Neuroinflammation2016ISSN 1742-2094
    Sayyad, Wasim A. et al.The role of Rac1 in the growth cone dynamics and force generation of DRG neuronsPLoS ONE2016ISSN 1932-6203
    Hamers, Anouk A.J. et al.Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and toleranceBMC Genomics2016ISSN 1471-2164
    Valdez, Chris M. et al.The Rac-GAP alpha2-chimaerin regulates hippocampal dendrite and spine morphogenesisMolecular and Cellular Neuroscience2016ISSN 1095-9327
    Lampi, Marsha C. et al.Simvastatin ameliorates matrix stiffness-mediated endothelial monolayer disruptionPLoS ONE2016ISSN 1932-6203
    Zhao, Chen Ze et al.Inhibition of farnesyl pyrophosphate synthase improves pressure overload induced chronic cardiac remodelingScientific Reports2016ISSN 2045-2322
    Sroka, Jolanta et al.Lamellipodia and membrane blebs drive efficient electrotactic migration of rat walker carcinosarcoma cells WC 256PLoS ONE2016ISSN 1932-6203
    Xiao, Bin et al.Extracellular translationally controlled tumor protein promotes colorectal cancer invasion and metastasis through Cdc42/JNK/ MMP9 signalingOncotarget2016ISSN 1949-2553
    Caution, Kyle et al.Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearanceScientific Reports2015ISSN 2045-2322
    Zeineddine, Rafaa et al.SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagationMolecular Neurodegeneration2015ISSN 1750-1326
    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 Microbiology2015ISSN 1664-302X
    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 Metabolism2015ISSN 1559-7016
    Herberich, Stefanie E. et al.ANKS1B Interacts with the cerebral cavernous malformation protein-1 and controls endothelial permeability but not sprouting angiogenesisPLoS ONE2015ISSN 1932-6203
    Guo, Yuna et al.A novel pharmacologic activity of ketorolac for therapeutic benefit in ovarian cancer patientsClinical Cancer Research2015ISSN 1557-3265
    Breslin, Jerome W. et al.Involvement of local lamellipodia in endothelial barrier functionPLoS ONE2015ISSN 1932-6203
    Ochoa-Alvarez, Jhon A. et al.Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanismsOncotarget2015ISSN 1949-2553
    Rigano, Luciano A. et al.Listeria monocytogenes antagonizes the human GTPase Cdc42 to promote bacterial spreadCellular Microbiology2014ISSN 1462-5822
    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 Immunology2014ISSN 0022--1767
    Marinković, Goran et al.6-mercaptopurine reduces macrophage activation and gut epithelium proliferation through inhibition of GTPase Rac1Inflammatory Bowel Diseases2014ISSN 1536-4844
    Mackay, Joanna L. et al.Simultaneous and independent tuning of RhoA and Rac1 activity with orthogonally inducible promotersIntegrative Biology (United Kingdom)2014ISSN 1757-9708
    Maurya, Vineet Kumar et al.Expression and activity of Rac1 is negatively affected in the dehydroepiandrosterone induced polycystic ovary of mouseJournal of Ovarian Research2014ISSN 1757-2215
    Aguilar, Helena et al.VAV3 mediates resistance to breast cancer endocrine therapyBreast Cancer Research2014ISSN 1465-542X
    Kalia, Manjula et al.Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic MechanismJournal of Virology2013ISSN 0022--538X
    Buranda, Tione et al.Rapid parallel flow cytometry assays of active GTPases using effector beadsAnalytical Biochemistry2013ISSN 1096-0309
    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 Genetics2013ISSN 0964-6906
    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 Pathology2013ISSN 0002-9440
    Yang, Jian et al.Cardiac-specific overexpression of farnesyl pyrophosphate synthase induces cardiac hypertrophy and dysfunction in miceCardiovascular Research2013ISSN 0008-6363
    Dubash, Adi D. et al.The GEF Bcr activates RhoA/MAL signaling to promote keratinocyte differentiation via desmoglein-1Journal of Cell Biology2013ISSN 0021-9525
    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 Biochemistry2013ISSN 1421-9778
    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 lenalidomideBlood2013ISSN 1528-0020
    Oshikawa, Jin et al.Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cellsAmerican Journal of Physiology - Heart and Circulatory Physiology2012ISSN 0363-6135
    Montalvo-Ortiz, Brenda L. et al.Characterization of EHop-016, novel small molecule inhibitor of Rac GTPaseThe Journal of biological chemistry2012ISSN 1083--351X
    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 physiology2012ISSN 1097--4652
    Stefanini, Lucia et al.Rap1-Rac1 circuits potentiate platelet activationArteriosclerosis, thrombosis, and vascular biology2012ISSN 1524--4636
    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 ErkOncogene2012ISSN 0950-9232
    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 Pathology2012ISSN 0002-9440
    Chen, Si Meng et al.Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTORBiochemical Pharmacology2012ISSN 0006-2952
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    Vives, Virginie et al.The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts2011ISSN 1523--4681

    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.