RhoA G-LISA Activation Assay (Luminescence format) 96 assays

G-LISA RhoA Activation Assay Biochem Kit (luminescence format)
$0.00

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
This G-LISA™ Rho activation assay measures the levels of GTP-loaded RhoA in cells. The level of activation is measured with luminometry. The G-LISA Rho activation assays are ELISA based Rho activation assays with wich you can measure Rho activity in cells in less than 3 h. For a more detailed introduction on G-LISA™ assays and a listing of other available G-LISA™ kits, see our main G-LISA™ page. For a kit to measure RhoA activation with colorimetric detection, see Cat. # BK124

 

Kit contents
The kit contains sufficient reagents to perform 96 Rho 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:

  1. 96 Rho-GTP affinity wells
  2. Lysis buffer
  3. Binding buffer
  4. Antigen presenting buffer
  5. Wash buffer
  6. Antibody dilution buffer
  7. Anti-RhoA antibody
  8. HRP-labeled secondary antibody
  9. Positive control RhoA protein
  10. Protease inhibitor cocktail (Cat. # PIC02)
  11. Luminescence detection reagents
  12. Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
  13. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

  1. Luminometer capable of reading a 96-well plate
  2. Multichannel or multidispensing pipettor
  3. Orbital microplate shaker capable of at least 200 rpm shaking (400 rpm is optimal)

Example results
Serum starved Swiss 3T3, HeLa and A431 cells were stimulated with the Rho activating compound lysophosphatidic acid and RhoA activation was measured with BK121 (Fig 1)

g-lisa_2

Figure 1. Rho activation by lysophosphatidic acid (LPA) measured by G-LISA™ kit BK121. Swiss 3T3 (mouse), A431 (human) and HeLa (human) cells were serum starved followed by stimulation by LPA.  25 µg of lysates were subjected to the G-LISA™ assay.  Data shown are relative luminescence units (RLU) over background signal (wells incubated with lysis buffer alone instead of cell lysates).  Numbers above LPA bars correspond to fold activation compared to the control serum starved samples.

Go to main G-LISA™ page

G-LISA Products:
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)

Associated Products:a
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

 

  •  G-LISA Activation Assay Technical Guide download here
  • For our G-LISA Data Analysis (Luminescence) Excel Template please download here.

Talamás-Lara, D. et al. Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins. J. Eukaryot. Microbiol. 67, (2020).

Algaber, A. et al. MicroRNA-340-5p inhibits colon cancer cell migration via targeting of RhoA. Sci. Rep. 10, 16934 (2020).

dos Santos, M. A. et al. Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cγ1. Immunobiology 225, 151904 (2020).

Simonetti, M. et al. The impact of Semaphorin 4C/Plexin-B2 signaling on fear memory via remodeling of neuronal and synaptic morphology. Mol. Psychiatry 1–23 (2019) doi:10.1038/s41380-019-0491-4.

Mei, J. et al. A DAAM1 3′-UTR SNP mutation regulates breast cancer metastasis through affecting miR-208a-5p-DAAM1-RhoA axis. Cancer Cell Int. 19, 1–12 (2019).

Itano, S. et al. Colchicine attenuates renal fibrosis in a murine unilateral ureteral obstruction model. Mol. Med. Rep. 15, 4169–4175 (2017).

Moniz et al., 2013. Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasiveness. Hum. Mol. Genet. 22, 84-95.

Nobe et al., 2012. Two distinct dysfunctions in diabetic mouse mesenteric artery contraction are caused by changes in the Rho A–Rho kinase signaling pathway. E. J. Pharmacol. 683, 217-225.

Wang et al., 2012. RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response. Cardiovascular Diabetology. 11, 7.

Zuo et al., 2012. Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells. J. Cell. Physiol. 227, 1399-1407.

Alvarez et al., 2010. Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels. British J of Pharmacology. 160 ,1326-37.

Heckman-Stoddard et al., 2009. Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research. 11 ,http://breast-cancer-research.com/content/11/4/R61.

Hammar et al., 2009. Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell Function. Endocrinology. 150, 2072-2079.

Chastre et al., 2009. TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB J. 23, 916–928.

Kinoshita et al., 2008. Mol. Biol. Cell. 19, 2289

Moniz et al., 2008. WNK2 modulates MEK1 activity through the Rho GTPase pathway. Cell Signal. 20, 1762-68.

Lesato et al., 2008. Tiotropium Bromide Attenuates Respiratory Syncytial Virus Replication in Epithelial Cells. Respiration. 76, 434-441.

Kinoshita et al., 2008. Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation. Mol. Biol. Cell. 19, 2289-2299.

Scott et al., 2007. J Invest Dermatol. 127, 668.

Schreibelt et al., 2007. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB J. 21, 3666-3676.

Tanaka et al., 2007. Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth. J. Biol. Chem. 282, 10506-10515.

Higashibata et al., 2006. BMC Biochem. 7, 19

Zuo et al., 2006. Biochem Biophys Res Commun. 351, 361

Woods and Beier, 2006. J Biol Chem. 281, 13134

AuthorTitleJournalYearArticle Link
Lee, Sang Joon et al.AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defenceNature2021ISSN 1476-4687
Simonetti, Manuela et al.The impact of Semaphorin 4C/Plexin-B2 signaling on fear memory via remodeling of neuronal and synaptic morphologyMolecular Psychiatry2021ISSN 1476-5578
Talamás-Lara, Daniel et al.Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding ProteinsThe Journal of eukaryotic microbiology2020ISSN 1550--7408
dos Santos, Marlus Alves et al.Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cγ1Immunobiology2020ISSN 1878--3279
Algaber, Anwar et al.MicroRNA-340-5p inhibits colon cancer cell migration via targeting of RhoAScientific Reports 2020 10:12020ISSN 2045--2322
Mei, Jie et al.A DAAM1 3′-UTR SNP mutation regulates breast cancer metastasis through affecting miR-208a-5p-DAAM1-RhoA axisCancer Cell International2019ISSN 1475-2867
Wu, Xuping et al.Wnt5a induces ROR1 and ROR2 to activate RhoA in esophageal squamous cell carcinoma cellsCancer Management and Research2019ISSN 1179-1322
Elshaer, Sally L. et al.Modulation of the p75 neurotrophin receptor using LM11A-31 prevents diabetes-induced retinal vascular permeability in mice via inhibition of inflammation and the RhoA kinase pathwayDiabetologia2019ISSN 1432-0428
Yan, Ting et al.Integrin αvβ3-associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cellsJournal of Biological Chemistry2018ISSN 1083-351X
Alizadeh, Javad et al.Detection of small gtpase prenylation and gtp binding using membrane fractionation and gtpase-linked immunosorbent assayJournal of Visualized Experiments2018ISSN 1940-087X
Itano, Seiji et al.Colchicine attenuates renal fibrosis in a murine unilateral ureteral obstruction modelMolecular Medicine Reports2017ISSN 1791-3004
Kim, Jongshin et al.YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturationJournal of Clinical Investigation2017ISSN 1558-8238
Ruggiero, Carmen et al.Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasionScience Signaling2017ISSN 1937-9145
Paldy, Eszter et al.Semaphorin 4C Plexin-B2 signaling in peripheral sensory neurons is pronociceptive in a model of inflammatory painNature Communications2017ISSN 2041-1723
López-Contreras, Luilli et al.Structural and functional characterization of the divergent Entamoeba Src using Src inhibitor-1Parasites and Vectors2017ISSN 1756-3305
Park, Yong Hwan et al.Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDSNature Immunology2016ISSN 1529-2916
Herrera-Martínez, Mayra et al.Antiamoebic activity of Adenophyllum aurantium (L.) strother and its effect on the actin cytoskeleton of Entamoeba histolyticaFrontiers in Pharmacology2016ISSN 1663-9812
Nour-Eldine, Wared et al.Adiponectin attenuates angiotensin II-induced vascular smooth muscle cell remodeling through nitric oxide and the RhoA/ROCK pathwayFrontiers in Pharmacology2016ISSN 1663-9812
Yan, Yi et al.Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5Nature Communications2015ISSN 2041-1723
Ajima, Rieko et al.DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assemblyDevelopmental Biology2015ISSN 1095-564X
Lasgorceix, M. et al.In vitro and in vivo evaluation of silicated hydroxyapatite and impact of insulin adsorptionJournal of Materials Science: Materials in Medicine2014ISSN 1573-4838
Nakajima, Chikako et al.The lipoprotein receptor LRP1 modulates sphingosine-1-phosphate signaling and is essential for vascular developmentDevelopment (Cambridge)2014ISSN 1477-9129
Moniz, Sónia et al.Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasivenessHuman molecular genetics2013ISSN 1460--2083
Herrera-Martínez, M. et al.Actin, RhoA, and Rab11 participation during encystment in entamoeba invadensBioMed Research International2013ISSN 2314-6133
Naraoka, Masato et al.Suppression of the Rho/Rho-Kinase Pathway and Prevention of Cerebral Vasospasm by Combination Treatment with Statin and Fasudil After Subarachnoid Hemorrhage in RabbitTranslational Stroke Research2013ISSN 1868-4483
Bai, Xue et al.The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertensionNature Communications2013ISSN 2041-1723
Veeramah, Krishna R. et al.Exome sequencing reveals new causal mutations in children with epileptic encephalopathiesEpilepsia2013ISSN 0013-9580
Bray, Kristi et al.Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migrationBreast Cancer Research2013ISSN 1465-5411
Wang, Jiping et al.RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular responseCardiovascular Diabetology2012ISSN 1475-2840
Nobe, Koji et al.Two distinct dysfunctions in diabetic mouse mesenteric artery contraction are caused by changes in the Rho A-Rho kinase signaling pathwayEuropean journal of pharmacology2012ISSN 1879--0712
Zuo, Yufeng et al.Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cellsJournal of cellular physiology2012ISSN 1097--4652
Doherty, Jason T. et al.Skeletal muscle differentiation and fusion are regulated by the BAR-containing Rho-GTPase-activating Protein (Rho-GAP), GRAFJournal of Biological Chemistry2011ISSN 0021-9258
Alvarez, S. M. et al.Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vesselsBritish journal of pharmacology2010ISSN 1476--5381
Heckman-Stoddard, Brandy M. et al.Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progressionBreast Cancer Research : BCR2009ISSN 1465-5411
Hammar, Eva et al.Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell FunctionEndocrinology2009ISSN 0013--7227
Chastre, Eric et al.TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasivenessThe FASEB Journal2009ISSN 1530--6860
Iesato, Ken et al.Tiotropium bromide attenuates respiratory syncytial virus replication in epithelial cellsRespiration; international review of thoracic diseases2008ISSN 1423--0356
Kinoshita, Nagatoki et al.Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube FormationMolecular Biology of the Cell2008ISSN 1059-1524
Kinoshita, Nagatoki et al.Apical accumulation of Rho in the neural plate is important for neural plate cell shape change and neural tube formationMolecular Biology of the Cell2008ISSN 1059-1524
Moniz, Sónia et al.WNK2 modulates MEK1 activity through the Rho GTPase pathwayCellular signalling2008ISSN 0898--6568
Scott, Glynis A. et al.Lysophosphatidylcholine mediates melanocyte dendricity through PKCzeta activationThe Journal of investigative dermatology2007ISSN 1523--1747
Schreibelt, Gerty et al.Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signalingFASEB journal : official publication of the Federation of American Societies for Experimental Biology2007ISSN 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 outgrowthThe Journal of biological chemistry2007ISSN 0021--9258
Higashibata, Akira et al.Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation - Molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factorBMC Biochemistry2006ISSN 1471-2091
Woods, Anita et al.RhoA/ROCK Signaling Regulates Chondrogenesis in a Context-dependent Manner *Journal of Biological Chemistry2006ISSN 0021--9258

Question 1:  There is less than a 2-fold difference in signal intensity between my positive control and lysis buffer blank.  Why?

Answer 1:  To accurately measure luminescence signal intensity between the positive control and buffer blank, please check the instrument settings on the luminometer (see below for suggestions).  We also recommend running some “set-up” experiments with just the buffer blank and positive control to determine optimal settings for detecting the positive control signal 3-5 fold higher than the buffer blank.  It is also important to remember to use a fresh control protein tube for each run of positive control samples.  Do not store and re-use the positive control.

Machine Settings

 Gain    

Gain controls the sensitivity of the machine.  Most luminometers do not allow manual alteration of gain and use an auto-calibration or limited calibration function.  Turn off auto-settings and auto-calibration to use the machine in manual mode.  It is important to contact the luminometer manufacturer or consult the user’s manual to determine the best way to alter the machine sensitivity.  If gain can be altered, one should read at low, medium and high gains to determine the reading within the linear range of the assay (positive control should be 3-5X higher than blank). Gain range varies with instrument.  For example, gain in the Tecan GmbH SpectroFluor Plus ranges from 0 - 150 (where 150 is the highest).

 Integration Time

This parameter can be varied on most machines.  It is a good idea to set the machine at the lowest integration time (usually 10 – 100 ms). Integration times greater than 200 ms are likely to read out of the linear range of the assay and may require lowering of gain or dilution of primary and/or secondary antibodies.

 Shaking

Most machines give the shaking option.  The recommended setting is 5 sec shake, medium orbital speed before read.  This option is not essential to the assay.

 Temperature

Room temperature

 Plate type

Any setting that specifies 96 well flat, white will be sufficient.

 Filters

Luminescence does not require excitation or emission filters so the filter spaces should be left blank.  If this is not an option, excitation can be set at any value and emission should be set between 400-500nm, with 430-445 as optimal setting.

 

Question 2:  My arbitrary luminescence units (ALU) or relative luminescence units (RLU) are very different from what is depicted in the manual.  Why?

Answer 2:  This is very typical as the luminescence units will vary from luminometer to luminometer based on the machine’s sensitivity and instrument settings.  The important information to take note of is what the relationship is between buffer blank and positive control luminescence values.  The positive control signal should be 3-5 fold greater than the buffer blank luminescence signal.  If that is the case, then the G-LISA assay is functioning in the linear range and experimental samples can now be processed.

 

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