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.
Figure 1.The simple procedure of the Rac1 G-LISA™ assay.
Kit Contents - Enough reagents for 96 assays (Stripwell)
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.
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|Francis, Caitlin R. et al.||Rab35 governs apicobasal polarity through regulation of actin dynamics during sprouting angiogenesis||Nature Communications 2022 13:1||2022||ISSN 2041--1723|
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|Harms, Julia et al.||Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion||Cancer Gene Therapy 2022||2022||ISSN 1476--5500|
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|Vallejo, Daniela et al.||Wnt5a modulates dendritic spine dynamics through the regulation of Cofilin via small Rho GTPase activity in hippocampal neurons||Journal of Neurochemistry||2021||ISSN 1471-4159|
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|Andrews, Madeline G. et al.||Mtor signaling regulates the morphology and migration of outer radial glia in developing human cortex||eLife||2020||ISSN 2050-084X|
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|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 Pathway||International Journal of Molecular Sciences||2020||ISSN 1422-0067|
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|Chen, Guo Ping et al.||Geranylgeranyl Transferase-I Knockout Inhibits Oxidative Injury of Vascular Smooth Muscle Cells and Attenuates Diabetes-Accelerated Atherosclerosis||Journal of Diabetes Research||2020||ISSN 2314-6753|
|Woida, Patrick J. et al.||The Vibrio cholerae MARTX toxin silences the inflammatory response to cytoskeletal damage before inducing actin cytoskeleton collapse||Science Signaling||2020||ISSN 1937-9145|
|Larribère, Lionel et al.||NF1-RAC1 axis regulates migration of the melanocytic lineage||Translational Oncology||2020||ISSN 1936-5233|
|Kowluru, Renu A.||Retinopathy in a diet-induced type 2 diabetic rat model and role of epigenetic modifications||Diabetes||2020||ISSN 1939-327X|
|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 formation||International Journal of Molecular Sciences||2020||ISSN 1422-0067|
|Krueger, Irena et al.||Reelin amplifies glycoprotein VI activation and alphaiib beta3 integrin outside-in signaling via PLC Gamma 2 and Rho GTPases||Arteriosclerosis, Thrombosis, and Vascular Biology||2020||ISSN 1524-4636|
|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 models||FASEB Journal||2020||ISSN 1530-6860|
|Zanin, Juan P. et al.||The p75NTR influences cerebellar circuit development and adult behavior via regulation of cell cycle duration of granule cell progenitors||Journal of Neuroscience||2019||ISSN 1529-2401|
|Shi, Wei et al.||EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells||Journal of Biological Chemistry||2019||ISSN 1083-351X|
|Gokoffski, Kimberly K. et al.||Physiologic electrical fields direct retinal ganglion cell axon growth in vitro||Investigative Ophthalmology and Visual Science||2019||ISSN 1552-5783|
|Wang, Baomei et al.||Macrophage β2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced Colitis||Cell Reports||2019||ISSN 2211-1247|
|Soni, Upendra Kumar et al.||A high level of TGF-B1 promotes endometriosis development via cell migration, adhesiveness, colonization, and invasiveness||Biology of Reproduction||2019||ISSN 1529-7268|
|Laufer, Julia M. et al.||Chemokine Receptor CCR7 Triggers an Endomembrane Signaling Complex for Spatial Rac Activation||Cell Reports||2019||ISSN 2211-1247|
|Stefanelli, Victoria L. et al.||Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion||Matrix Biology||2019||ISSN 1569-1802|
|Wu, Wenjuan et al.||Inhibition of Rac1-dependent forgetting alleviates memory deficits in animal models of Alzheimer’s disease||Protein and Cell||2019||ISSN 1674-8018|
|Haq, Naila et al.||Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons||PLoS Biology||2019||ISSN 1545-7885|
|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 KRas||Cell Death and Disease||2019||ISSN 2041-4889|
|Saito-Reis, Chelsea A. et al.||The tetraspanin CD82 regulates bone marrow homing and engraftment of hematopoietic stem and progenitor cells||Molecular Biology of the Cell||2018||ISSN 1939-4586|
|Rachubik, Patrycja et al.||The TRPC6-AMPK pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytes||Cellular Physiology and Biochemistry||2018||ISSN 1421-9778|
|Santhana Kumar, Karthiga et al.||TGF-β Determines the Pro-migratory Potential of bFGF Signaling in Medulloblastoma||Cell Reports||2018||ISSN 2211-1247|
|Suraneni, Praveen K. et al.||Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity||Blood Advances||2018||ISSN 2473-9537|
|Caggia, Silvia et al.||Novel role of Giα2 in cell migration: Downstream of PI3-kinase–AKT and Rac1 in prostate cancer cells||Journal of Cellular Physiology||2018||ISSN 1097-4652|
|Duraisamy, Arul J. et al.||Epigenetics and regulation of oxidative stress in diabetic retinopathy||Investigative Ophthalmology and Visual Science||2018||ISSN 1552-5783|
|Olabi, Safiah et al.||Integrin-Rac signalling for mammary epithelial stem cell self-renewal 06 Biological Sciences 0601 Biochemistry and Cell Biology||Breast Cancer Research||2018||ISSN 1465-542X|
|Mayer, Louisa et al.||Nbeal2 interacts with Dock7, Sec16a, and Vac14||Blood||2018||ISSN 1528-0020|
|Stierwalt, Harrison D. et al.||Insulin-stimulated Rac1-GTP binding is not impaired by palmitate treatment in L6 myotubes||Physiological Reports||2018||ISSN 2051-817X|
|Proto, Jonathan D. et al.||Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation Resolution||Immunity||2018||ISSN 1097-4180|
|Dyberg, Cecilia et al.||Rho-associated kinase is a therapeutic target in neuroblastoma||Proceedings of the National Academy of Sciences of the United States of America||2017||ISSN 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 Rac1||Journal of Surgical Research||2017||ISSN 1095-8673|
|Moreno-Layseca, Paulina et al.||The requirement of integrins for breast epithelial proliferation||European Journal of Cell Biology||2017||ISSN 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 implantation||Scientific Reports||2017||ISSN 2045-2322|
|Ubba, Vaibhave et al.||RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model||Reproductive Sciences||2017||ISSN 1933-7205|
|Chen, Guo Ping et al.||Inhibition of farnesyl pyrophosphate synthase attenuates high glucose-induced vascular smooth muscle cells proliferation||Molecular Medicine Reports||2017||ISSN 1791-3004|
|Gu, Changkyu et al.||Dynamin autonomously regulates podocyte focal adhesion maturation||Journal of the American Society of Nephrology||2017||ISSN 1533-3450|
|Baranov, Maksim V. et al.||SWAP70 Organizes the Actin Cytoskeleton and Is Essential for Phagocytosis||Cell Reports||2016||ISSN 2211-1247|
|Valdez, Chris M. et al.||The Rac-GAP alpha2-chimaerin regulates hippocampal dendrite and spine morphogenesis||Molecular and Cellular Neuroscience||2016||ISSN 1095-9327|
|Sayyad, Wasim A. et al.||The role of Rac1 in the growth cone dynamics and force generation of DRG neurons||PLoS ONE||2016||ISSN 1932-6203|
|Lampi, Marsha C. et al.||Simvastatin ameliorates matrix stiffness-mediated endothelial monolayer disruption||PLoS ONE||2016||ISSN 1932-6203|
|Jones, Eleanor L. et al.||Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the Tetraspanins CD82 and CD37||The Journal of Immunology||2016||ISSN 0022--1767|
|Zhao, Chen Ze et al.||Inhibition of farnesyl pyrophosphate synthase improves pressure overload induced chronic cardiac remodeling||Scientific Reports||2016||ISSN 2045-2322|
|Sroka, Jolanta et al.||Lamellipodia and membrane blebs drive efficient electrotactic migration of rat walker carcinosarcoma cells WC 256||PLoS ONE||2016||ISSN 1932-6203|
|Rom, Slava et al.||PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrier||Journal of Neuroinflammation||2016||ISSN 1742-2094|
|Hamers, Anouk A.J. et al.||Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance||BMC Genomics||2016||ISSN 1471-2164|
|Xiao, Bin et al.||Extracellular translationally controlled tumor protein promotes colorectal cancer invasion and metastasis through Cdc42/JNK/ MMP9 signaling||Oncotarget||2016||ISSN 1949-2553|
|Herberich, Stefanie E. et al.||ANKS1B Interacts with the cerebral cavernous malformation protein-1 and controls endothelial permeability but not sprouting angiogenesis||PLoS ONE||2015||ISSN 1932-6203|
|Caution, Kyle et al.||Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance||Scientific Reports||2015||ISSN 2045-2322|
|Ochoa-Alvarez, Jhon A. et al.||Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms||Oncotarget||2015||ISSN 1949-2553|
|Rom, Slava et al.||Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditions||Journal of Cerebral Blood Flow and Metabolism||2015||ISSN 1559-7016|
|Zeineddine, Rafaa et al.||SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation||Molecular Neurodegeneration||2015||ISSN 1750-1326|
|Guo, Yuna et al.||A novel pharmacologic activity of ketorolac for therapeutic benefit in ovarian cancer patients||Clinical Cancer Research||2015||ISSN 1557-3265|
|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 activity||Frontiers in Microbiology||2015||ISSN 1664-302X|
|Breslin, Jerome W. et al.||Involvement of local lamellipodia in endothelial barrier function||PLoS ONE||2015||ISSN 1932-6203|
|Rigano, Luciano A. et al.||Listeria monocytogenes antagonizes the human GTPase Cdc42 to promote bacterial spread||Cellular Microbiology||2014||ISSN 1462-5822|
|Maurya, Vineet Kumar et al.||Expression and activity of Rac1 is negatively affected in the dehydroepiandrosterone induced polycystic ovary of mouse||Journal of Ovarian Research||2014||ISSN 1757-2215|
|Marinković, Goran et al.||Inhibition of GTPase Rac1 in Endothelium by 6-Mercaptopurine Results in Immunosuppression in Nonimmune Cells: New Target for an Old Drug||The Journal of Immunology||2014||ISSN 0022--1767|
|Mackay, Joanna L. et al.||Simultaneous and independent tuning of RhoA and Rac1 activity with orthogonally inducible promoters||Integrative Biology (United Kingdom)||2014||ISSN 1757-9708|
|Aguilar, Helena et al.||VAV3 mediates resistance to breast cancer endocrine therapy||Breast Cancer Research||2014||ISSN 1465-542X|
|Marinković, Goran et al.||6-mercaptopurine reduces macrophage activation and gut epithelium proliferation through inhibition of GTPase Rac1||Inflammatory Bowel Diseases||2014||ISSN 1536-4844|
|Dubash, Adi D. et al.||The GEF Bcr activates RhoA/MAL signaling to promote keratinocyte differentiation via desmoglein-1||Journal of Cell Biology||2013||ISSN 0021-9525|
|Yang, Jian et al.||Cardiac-specific overexpression of farnesyl pyrophosphate synthase induces cardiac hypertrophy and dysfunction in mice||Cardiovascular Research||2013||ISSN 0008-6363|
|Buranda, Tione et al.||Rapid parallel flow cytometry assays of active GTPases using effector beads||Analytical Biochemistry||2013||ISSN 1096-0309|
|Kalia, Manjula et al.||Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism||Journal of Virology||2013||ISSN 0022--538X|
|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-β||Human Molecular Genetics||2013||ISSN 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 barrier||American Journal of Pathology||2013||ISSN 0002-9440|
|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******||Blood||2013||ISSN 1528-0020|
|Chen, Bin et al.||Alteration of mevalonate pathway related enzyme expressions in pressure overload-induced cardiac hypertrophy and associated heart failure with preserved ejection fraction||Cellular Physiology and Biochemistry||2013||ISSN 1421-9778|
|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) 2||Journal of Biological Chemistry||2012||ISSN 0021-9258|
|Sánchez, Elena G. et al.||African swine fever virus uses macropinocytosis to enter host cells||PLoS Pathogens||2012||ISSN 1553-7374|
|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 90||Journal of cellular physiology||2012||ISSN 1097--4652|
|Greco, Carolina M. et al.||Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor||Cardiovascular Research||2012||ISSN 0008--6363|
|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 cancer||Blood||2012||ISSN 1528-0020|
|Oshikawa, Jin et al.||Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells||American Journal of Physiology - Heart and Circulatory Physiology||2012||ISSN 0363-6135|
|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 Erk||Oncogene||2012||ISSN 0950-9232|
|Montalvo-Ortiz, Brenda L. et al.||Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase||The Journal of biological chemistry||2012||ISSN 1083--351X|
|Chen, Si Meng et al.||Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR||Biochemical Pharmacology||2012||ISSN 0006-2952|
|Stefanini, Lucia et al.||Rap1-Rac1 circuits potentiate platelet activation||Arteriosclerosis, thrombosis, and vascular biology||2012||ISSN 1524--4636|
|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 conformation||American Journal of Pathology||2012||ISSN 0002-9440|
|Vives, Virginie et al.||The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts||2011||ISSN 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.
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.
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