RhoA G-LISA Activation Assay Kit (Colorimetric format) 96 assays

G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)

$0.00

SKU

BK124

Datasheet

MSDS

Amount / Package

96 assays (Cat.# BK124) + $968.00

24 assays (Cat. # BK124-S) + $584.00

Save 10%: Active + Total RhoA Assay: 96 assays (Cat.# BK124) + 96 assays (Cat.# BK150) + $1,346.40

Bundle Offer! RhoA/Rac1/Cdc42 G-LISA 3 Kits (24 assays per kit) (Cat. # BK135) + $1,613.00

Qty:

Details

Product Uses Include

Rho signaling pathway studies
Rho activation assays with primary cells
Studies of Rho activators and inactivators
Rho activation assays with limited material
High throughput screens for Rho activation

Introduction
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.

Click Here for Detailed Methods

Kit contents
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:

Rho-GTP affinity wells (12 strips of 8 wells)
Lysis buffer
Binding buffer
Antigen presenting buffer
Wash buffer
Antibody dilution buffer
Anti-RhoA antibody
HRP-labeled secondary antibody
Positive control RhoA protein
Protease inhibitor cocktail (Cat. # PIC02)
Absorbance detection reagents
Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

96-well plate spectrophotometer capable of reading 490 nm wavelength
Multichannel or multidispensing pipettor
Orbital microplate shaker capable of at least 200 rpm shaking (400 rpm is optimal)

Example results
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.

About

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

G-LISA Activation Assay Technical Guide download here
G-LISA Data Analysis (Absorbance) Excel Template download here.

Citations

Author	Title	Journal	Year	Article Link
Anastasaki, Corina et al.	Generation of human induced pluripotent stem cell-derived cerebral organoids for cellular and molecular characterization	STAR Protocols	2022	ISSN 2666--1667
Vadakumchery, Anila et al.	The Small GTPase RHOA Links SLP65 Activation to PTEN Function in Pre B Cells and Is Essential for the Generation and Survival of Normal and Malignant B Cells	Frontiers in Immunology	2022	ISSN 1664-3224
Zanin, Juan P. et al.	p75NTR prevents the onset of cerebellar granule cell migration via RhoA activation	eLife	2022	ISSN 2050-084X
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
Kumar, Akhilesh et al.	Essential role of Rnd1 in innate immunity during viral and bacterial infections	Cell Death & Disease	2022	Article Link
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
Xue, Tan et al.	Effects of Aster B-mediated intracellular accumulation of cholesterol on inflammatory process and myocardial cells in acute myocardial infarction	Hellenic Journal of Cardiology	2022
Hauke, Michael et al.	Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells	Journal of the American Heart Association	2022	ISSN 2047-9980
Darp, Revati et al.	Oncogenic BRAF induces whole-genome doubling through suppression of cytokinesis	Nature Communications 2022 13:1	2022	ISSN 2041--1723
Meng, Zhipeng et al.	The Hippo pathway mediates Semaphorin signaling	Science Advances	2022	ISSN 2375-2548
de Vallière, Cheryl et al.	pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis	International Journal of Molecular Sciences	2022	ISSN 1422-0067
Wang, Kankai et al.	PTBP1 knockdown promotes neural differentiation of glioblastoma cells through UNC5B receptor	Theranostics	2022	ISSN 1838-7640
Kumar, Akhilesh et al.	Essential role of Rnd1 in innate immunity during viral and bacterial infections	Cell Death & Disease 2022 13:6	2022	ISSN 2041--4889
Yadav, Vikas et al.	Increased MARCKS Activity in BRAF Inhibitor-Resistant Melanoma Cells Is Essential for Their Enhanced Metastatic Behavior Independent of Elevated WNT5A and IL-6 Signaling	Cancers	2022	ISSN 2072-6694
Weder, Bruce et al.	New Therapeutic Approach for Intestinal Fibrosis Through Inhibition of pH-Sensing Receptor GPR4	Inflammatory Bowel Diseases	2022	ISSN 1078--0998
Sánchez-de la Torre, Aníbal et al.	Cannabinoid CB1 receptor gene inactivation in oligodendrocyte precursors disrupts oligodendrogenesis and myelination in mice	Cell Death & Disease 2022 13:7	2022	ISSN 2041--4889
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
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
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
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
Shoda, Tetsuo et al.	Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis	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
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
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
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
Bhosle, Vikrant K. et al.	SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling	Nature Communications	2020	ISSN 2041-1723
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
Dias Gomes, Martim et al.	Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity	Nature Communications	2019	ISSN 2041-1723
Majolée, Jisca et al.	CSN5 inhibition triggers inflammatory signaling and Rho/ROCK-dependent loss of endothelial integrity	Scientific Reports	2019	ISSN 2045-2322
Santhana Kumar, Karthiga et al.	TGF-β Determines the Pro-migratory Potential of bFGF Signaling in Medulloblastoma	Cell Reports	2018	ISSN 2211-1247
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
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
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
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
López-Posadas, Rocío et al.	Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation	Journal of Clinical Investigation	2016	ISSN 1558-8238
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
Patra, Vijay Kumar et al.	The Skin Microbiome: Is It Affected by UV-induced Immune Suppression?	Frontiers in Microbiology	2016	ISSN 1664-302X
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Tan, Hong et al.	Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves	Developmental Biology	2013	ISSN 1095-564X
Kalia, Manjula et al.	Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism	Journal of Virology	2013	ISSN 0022--538X
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
Zhou, Zhigang et al.	HSV-mediated gene transfer of C3 transferase inhibits Rho to promote axonal regeneration	Experimental Neurology	2012	ISSN 0014--4886
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
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
Takefuji, Mikito et al.	G13-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure	Circulation	2012	ISSN 0009-7322
Howe, Grant A. et al.	RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA	Vascular Cell	2012	ISSN 2045--824X
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
Yang, Seungwon et al.	The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior	Biomaterials	2012	ISSN 1878--5905
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
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
Dhaliwal, Anandika et al.	Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases		2012	PMID 22509380
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
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
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
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	ISSN 0892--6638
Ganguly, Riya et al.	Adiponectin Increases LPL Activity via RhoA/ROCK-Mediated Actin Remodelling in Adult Rat Cardiomyocytes	Endocrinology	2011	ISSN 0013--7227
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
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
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
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
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
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
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
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
Chastre, Eric et al.	TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness	The FASEB Journal	2009	ISSN 1530--6860
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
Seifert, Jennifer L. et al.	Differential activation of Rac1 and RhoA in neuroblastoma cell fractions	Neuroscience letters	2009	ISSN 0304--3940
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.	Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation	Molecular Biology of the Cell	2008	ISSN 1059-1524
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
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
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
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
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
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

Faqs

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.