Product Uses Include
The G-LISA Rho activation assays are ELISA based Rho activation assays with which you can measure Rho activity in cells in less than 3 h. BK124 is very sensitive and has excellent accuracy between duplicate samples. For a more detailed introduction on G-LISA assays and a listing of other available G-LISA kits, see our main G-LISA page. The BK124 Rho activation assay kit measures the level of GTP-loaded RhoA only in cells. The level of activation is measured with absorbance set at 490nm. For a kit to measure RhoA activation with luminescence detection, see Cat. # BK121.
See G-LISA FAQs tab on our G-LISA page for more details.
The kit contains sufficient reagents to perform 96 RhoA activation assays. Since the Rho-GTP affinity wells are supplied as strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from one to multiple assays. The following components are included in the kit:
Serum starved Swiss 3T3 cells were stimulated with the Rho activating compound calpeptin and RhoA activation was measured with the G-LISA method (Figures 1 and 2)
Figure 1. RhoA activation by calpeptin measured by G-LISA kit BK124. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with calpeptin (Cal; 0.1 mg/ml for 30 min) or DMSO carrier only (SS). 10 µg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm.
Figure 2. Rho activity measured in Swiss 3T3 cells treated with the Cell Permeable Rho Inhibitor (CT04) using the RhoA G-LISA Activation Assay (Cat.# BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 0.20, 0.50 and 2.0 µg/ml of CT04 for 4h in serum free medium at 37°C, then activated with 100µg/ml calpeptin for 10min. Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124). Note: At 2.0 µg/ml CT04 for 4h results in almost complete (90%) inhibition of RhoA activity.
|Nath, Anu S. et al.||Modulation of the cell membrane lipid milieu by peroxisomal β-oxidation induces Rho1 signaling to trigger inflammatory responses||Cell Reports||2022||ISSN 2211-1247|
|Swaminathan, Bhairavi et al.||Endothelial Notch signaling directly regulates the small GTPase RND1 to facilitate Notch suppression of endothelial migration||Scientific Reports||2022||ISSN 2045-2322|
|Ma, Yuanyuan et al.||Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development||Development (Cambridge)||2021||ISSN 1477-9129|
|Shoda, Tetsuo et al.||Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis||Nature Communications||2021||ISSN 2041-1723|
|Jozic, Ivan et al.||Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds||Communications Biology||2021||ISSN 2399-3642|
|Mao, De Yu et al.||CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function||Science Signaling||2021||ISSN 1937-9145|
|Zhou, Qun et al.||Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability||Frontiers in Pharmacology||2021||ISSN 1663-9812|
|Gurusamy, Malarvizhi et al.||G-protein-coupled receptor P2Y10 facilitates chemokine-induced CD4 T cell migration through autocrine/paracrine mediators||Nature Communications||2021||ISSN 2041-1723|
|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|
|Porter, Lauren et al.||SUN1/2 Are Essential for RhoA/ROCK-Regulated Actomyosin Activity in Isolated Vascular Smooth Muscle Cells||Cells||2020||ISSN 2073-4409|
|Bhosle, Vikrant K. et al.||SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling||Nature Communications||2020||ISSN 2041-1723|
|Lachowski, Dariusz et al.||G Protein-Coupled Estrogen Receptor Regulates Actin Cytoskeleton Dynamics to Impair Cell Polarization||Frontiers in Cell and Developmental Biology||2020||ISSN 2296-634X|
|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|
|Salgado-Lucio, Monica L. et al.||FAK regulates actin polymerization during sperm capacitation via the ERK2/GEF-H1/RhoA signaling pathway||Journal of Cell Science||2020||ISSN 1477-9137|
|Fox, Megan E. et al.||Dendritic remodeling of D1 neurons by RhoA/Rho-kinase mediates depression-like behavior||Molecular Psychiatry||2020||ISSN 1476-5578|
|Hasan, Wan Nuraini Wan et al.||Annatto-derived tocotrienol promotes mineralization of MC3T3-E1 cells by enhancing BMP-2 protein expression via inhibiting RhoA activation and HMG-CoA reductase gene expression||Drug Design, Development and Therapy||2020||ISSN 1177-8881|
|Majolée, Jisca et al.||CSN5 inhibition triggers inflammatory signaling and Rho/ROCK-dependent loss of endothelial integrity||Scientific Reports||2019||ISSN 2045-2322|
|Dias Gomes, Martim et al.||Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity||Nature Communications||2019||ISSN 2041-1723|
|Sun, Xiao Qing et al.||Total flavones of Rhododendron simsii Planch flower protect isolated rat heart from ischaemia-reperfusion injury and its mechanism of UTR-RhoA-ROCK pathway inhibition||Journal of Pharmacy and Pharmacology||2018||ISSN 2042-7158|
|Tagashira, Toru et al.||Afadin facilitates vascular endothelial growth factor-induced network formation and migration of vascular endothelial cells by inactivating Rho-associated kinase through ARHGAP29||Arteriosclerosis, Thrombosis, and Vascular Biology||2018||ISSN 1524-4636|
|Ng, Tze Hann et al.||The Rho signalling pathway mediates the pathogenicity of AHPND-causing V. parahaemolyticus in shrimp||Cellular Microbiology||2018||ISSN 1462-5822|
|Li, Xu et al.||A positive feedback loop of profilin-1 and RhoA/ROCK1 promotes endothelial dysfunction and oxidative stress||Oxidative Medicine and Cellular Longevity||2018||ISSN 1942-0994|
|Santhana Kumar, Karthiga et al.||TGF-β Determines the Pro-migratory Potential of bFGF Signaling in Medulloblastoma||Cell Reports||2018||ISSN 2211-1247|
|Vautrin-Glabik, Alexia et al.||IP3R3 silencing induced actin cytoskeletal reorganization through ARHGAP18/RhoA/mDia1/FAK pathway in breast cancer cell lines||Biochimica et Biophysica Acta - Molecular Cell Research||2018||ISSN 1879-2596|
|Kapfhamer, Joshua et al.||The Gα q/11 -provoked induction of Akr1c18 in murine luteal cells is mediated by phospholipase C||Molecular and Cellular Endocrinology||2018||ISSN 1872-8057|
|Schillaci, Odessa et al.||Exosomes from metastatic cancer cells transfer amoeboid phenotype to non-metastatic cells and increase endothelial permeability: Their emerging role in tumor heterogeneity||Scientific Reports||2017||ISSN 2045-2322|
|Jackson, William D. et al.||Very-low and low-density lipoproteins induce neutral lipid accumulation and impair migration in monocyte subsets||Scientific Reports||2016||ISSN 2045-2322|
|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|
|Patra, Vijay Kumar et al.||The Skin Microbiome: Is It Affected by UV-induced Immune Suppression?||Frontiers in Microbiology||2016||ISSN 1664-302X|
|Yu, Yonghao et al.||Hydrogen-rich medium ameliorates lipopolysaccharide-induced barrier dysfunction via rhoa-mdia1 signaling in caco-2 cells||Shock||2016||ISSN 1540-0514|
|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|
|López-Posadas, Rocío et al.||Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation||Journal of Clinical Investigation||2016||ISSN 1558-8238|
|Lu, Wen Juan et al.||Senescence Mediated by p16INK4a Impedes Reprogramming of Human Corneal Endothelial Cells into Neural Crest Progenitors||Scientific Reports||2016||ISSN 2045-2322|
|Manukyan, Arkadi et al.||A complex of p190RhoGAP-A and anillin modulates RhoA-GTP and the cytokinetic furrow in human cells||Journal of Cell Science||2015||ISSN 1477-9137|
|Aifuwa, Ivie et al.||Senescent stromal cells induce cancer cell migration via inhibition of RhoA/ROCK/myosin-based cell contractility||Oncotarget||2015||ISSN 1949-2553|
|Cascio, Graciela et al.||CXCL12 Regulates through JAK1 and JAK2 Formation of Productive Immunological Synapses||The Journal of Immunology||2015||ISSN 0022--1767|
|Tang, Xiao et al.||HCLOCK Causes Rho-Kinase-Mediated Endothelial Dysfunction and NF-κ B-Mediated Inflammatory Responses||Oxidative Medicine and Cellular Longevity||2015||ISSN 1942-0994|
|Freeman, Spencer A. et al.||Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor||Nature Communications||2015||ISSN 2041-1723|
|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|
|Skrbic, Biljana et al.||Lack of collagen VIII reduces fibrosis and promotes early mortality and cardiac dilatation in pressure overload in mice||Cardiovascular Research||2015||ISSN 1755-3245|
|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|
|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|
|Ahn, Bum Ju et al.||Ninjurin1 enhances the basal motility and transendothelial migration of immune cells by inducing protrusive membrane dynamics||Journal of Biological Chemistry||2014||ISSN 1083-351X|
|Chen, Xiaofei et al.||The TMEFF2 tumor suppressor modulates integrin expression, RhoA activation and migration of prostate cancer cells||Biochimica et Biophysica Acta - Molecular Cell Research||2014||ISSN 1879-2596|
|Biechler V., Stefanie V. et al.||The impact of flow-induced forces on the morphogenesis of the outflow tract||Frontiers in Physiology||2014||ISSN 1664-042X|
|Beveridge, Ryan D. et al.||The leukemia-associated Rho guanine nucleotide exchange factor LARG is required for efficient replication stress signaling||Cell Cycle||2014||ISSN 1551-4005|
|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|
|Herr, Michael J. et al.||Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cells||PLoS ONE||2014||ISSN 1932-6203|
|Zhang, Di et al.||Co-expression of delta-catenin and RhoA is significantly associated with a malignant lung cancer phenotype||International Journal of Clinical and Experimental Pathology||2014||ISSN 1936-2625|
|Zhu, Ying Ting et al.||Knockdown of both p120 catenin and kaiso promotes expansion of human corneal endothelial monolayers via rhoa-rock-noncanonical BMP-NFκB pathway||Investigative Ophthalmology and Visual Science||2014||ISSN 1552-5783|
|Yang, Jian et al.||Inhibition of farnesyl pyrophosphate synthase attenuates angiotensin II-induced cardiac hypertrophy and fibrosis in vivo||International Journal of Biochemistry and Cell Biology||2013||ISSN 1357-2725|
|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|
|Tan, Hong et al.||Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves||Developmental Biology||2013||ISSN 1095-564X|
|Kanazawa, Yasushi et al.||The Rho-kinase inhibitor fasudil restores normal motor nerve conduction velocity in diabetic rats by assuring the proper localization of adhesion-related molecules in myelinating Schwann cells||Experimental neurology||2013||ISSN 1090--2430|
|DiScipio, Richard G. et al.||Complement C3a signaling mediates production of angiogenic factors in mesenchymal stem cells||Journal of Biomedical Science and Engineering||2013||ISSN 1937--6871|
|Kalia, Manjula et al.||Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism||Journal of Virology||2013||ISSN 0022--538X|
|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|
|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|
|Elali, Ayman et al.||Liver X receptor activation enhances blood-brain barrier integrity in the ischemic brain and increases the abundance of ATP-binding cassette transporters ABCB1 and ABCC1 on brain capillary cells||Brain Pathology||2012||ISSN 1015-6305|
|Zhu, Ying Ting et al.||Nuclear p120 catenin unlocks mitotic block of contactinhibited human corneal endothelial monolayers without disrupting adherent junctions||Journal of Cell Science||2012||ISSN 0021-9533|
|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|
|Takefuji, Mikito et al.||G13-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure||Circulation||2012||ISSN 0009-7322|
|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|
|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|
|Dhaliwal, Anandika et al.||Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases||2012||PMID 22509380|
|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|
|Ramseyer, Vanesa D. et al.||Tumor necrosis factor α decreases nitric oxide synthase type 3 expression primarily via Rho/Rho kinase in the thick ascending limb||Hypertension (Dallas, Tex. : 1979)||2012||ISSN 1524--4563|
|McCoy, Kelly L. et al.||Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop||Cellular signalling||2012||ISSN 1873--3913|
|Zhou, Zhigang et al.||HSV-mediated gene transfer of C3 transferase inhibits Rho to promote axonal regeneration||Experimental Neurology||2012|
|Garrido‐Gómez, Tamara et al.||Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F‐actin regulation||The FASEB Journal||2012|
|Yang, Seungwon et al.||The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior||Biomaterials||2012||ISSN 1878--5905|
|Howe, Grant A. et al.||RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA||Vascular Cell||2012||ISSN 2045--824X|
|Jin, Wanzhu et al.||Increased SRF transcriptional activity in human and mouse skeletal muscle is a signature of insulin resistance||Journal of Clinical Investigation||2011||ISSN 0021-9738|
|Mammoto, Tadanori et al.||Mechanochemical Control of Mesenchymal Condensation and Embryonic Tooth Organ Formation||Developmental Cell||2011||ISSN 1534-5807|
|Aguilar, Hector N. et al.||Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes||PLoS ONE||2011||ISSN 1932-6203|
|Ganguly, Riya et al.||Adiponectin Increases LPL Activity via RhoA/ROCK-Mediated Actin Remodelling in Adult Rat Cardiomyocytes||Endocrinology||2011||ISSN 0013--7227|
|Musso, Alessandra et al.||Relevance of the mevalonate biosynthetic pathway in the regulation of bone marrow mesenchymal stromal cell-mediated effects on T-cell proliferation and B-cell survival||Haematologica||2011||ISSN 1592--8721|
|Lichtenstein, Mathieu P. et al.||Secretase-independent and RhoGTPase/PAK/ERK-dependent regulation of cytoskeleton dynamics in astrocytes by NSAIDs and derivatives||Journal of Alzheimer's disease : JAD||2010||ISSN 1875--8908|
|Nini, Lylia et al.||Accurate and reproducible measurements of RhoA activation in small samples of primary cells||Analytical biochemistry||2010||ISSN 1096--0309|
|Romero, Ana M. et al.||Chronic ethanol exposure alters the levels, assembly, and cellular organization of the actin cytoskeleton and microtubules in hippocampal neurons in primary culture||Toxicological Sciences||2010||ISSN 1096-6080|
|Yang, Enyue et al.||Fluoride induces vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aortas||Environmental toxicology and pharmacology||2010||ISSN 1872--7077|
|Rapier, Rebecca et al.||The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells||Cancer Cell International||2010||ISSN 1475-2867|
|Rebillard, Amélie et al.||Cispl****-induced apoptosis involves a Fas-ROCK-ezrin-dependent actin remodelling in human colon cancer cells||European Journal of Cancer||2010||ISSN 0959-8049|
|Ridgway, Lon D. et al.||Modulation of GEF-H1 Induced Signaling by Heparanase in Brain Metastatic Melanoma Cells||Journal of cellular biochemistry||2010||ISSN 0730-2312|
|Chastre, Eric et al.||TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness||The FASEB Journal||2009||ISSN 1530--6860|
|Seifert, Jennifer L. et al.||Differential activation of Rac1 and RhoA in neuroblastoma cell fractions||Neuroscience letters||2009||ISSN 0304--3940|
|Hammar, Eva et al.||Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell Function||Endocrinology||2009||ISSN 0013--7227|
|Liu, Yang et al.||Ablation of p120-catenin enhances invasion and metastasis of human lung cancer cells||Cancer Science||2009||ISSN 1347-9032|
|Gien, Jason et al.||Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro||American Journal of Physiology - Lung Cellular and Molecular Physiology||2008||ISSN 1040-0605|
|Korobova, Farida et al.||Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells||Molecular biology of the cell||2008||ISSN 1939--4586|
|Moore, Simon W. et al.||Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1||Development||2008||ISSN 0950--1991|
|Sequeira, Linda et al.||Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis||Clinical & experimental metastasis||2008||ISSN 0262--0898|
|Mercer, Jason et al.||Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells||Science (New York, N.Y.)||2008||ISSN 1095--9203|
|Kinoshita, Nagatoki et al.||No Title||Molecular Biology of the Cell||2008||ISSN 1059-1524|
|Ramirez, Servio H. et al.||Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes||The Journal of Immunology||2008||ISSN 0022--1767|
|Rupp, Paul A. et al.||A role for RhoA in the two-phase migratory pattern of post-otic neural crest cells||Developmental Biology||2007||ISSN 0012-1606|
|Keely, Patricia J. et al.||Investigating integrin regulation and signaling events in three-dimensional systems||Methods in enzymology||2007||ISSN 0076--6879|
|Schreibelt, Gerty et al.||Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling||FASEB journal : official publication of the Federation of American Societies for Experimental Biology||2007||ISSN 1530--6860|
|Tanaka, Shigeru et al.||Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth||The Journal of biological chemistry||2007||ISSN 0021--9258|
|Bradley, William D. et al.||Integrin Signaling through Arg Activates p190RhoGAP by Promoting Its Binding to p120RasGAP and Recruitment to the Membrane||Molecular Biology of the Cell||2006||ISSN 1059-1524|
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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