Product Uses Include
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
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:
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)
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.
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)
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 firstname.lastname@example.org
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
|Lee, Sang Joon et al.||AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence||Nature||2021||ISSN 1476-4687|
|Simonetti, Manuela et al.||The impact of Semaphorin 4C/Plexin-B2 signaling on fear memory via remodeling of neuronal and synaptic morphology||Molecular Psychiatry||2021||ISSN 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 Proteins||The Journal of eukaryotic microbiology||2020||ISSN 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 Cγ1||Immunobiology||2020||ISSN 1878--3279|
|Algaber, Anwar et al.||MicroRNA-340-5p inhibits colon cancer cell migration via targeting of RhoA||Scientific Reports 2020 10:1||2020||ISSN 2045--2322|
|Mei, Jie et al.||A DAAM1 3′-UTR SNP mutation regulates breast cancer metastasis through affecting miR-208a-5p-DAAM1-RhoA axis||Cancer Cell International||2019||ISSN 1475-2867|
|Wu, Xuping et al.||Wnt5a induces ROR1 and ROR2 to activate RhoA in esophageal squamous cell carcinoma cells||Cancer Management and Research||2019||ISSN 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 pathway||Diabetologia||2019||ISSN 1432-0428|
|Yan, Ting et al.||Integrin αvβ3-associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells||Journal of Biological Chemistry||2018||ISSN 1083-351X|
|Alizadeh, Javad et al.||Detection of small gtpase prenylation and gtp binding using membrane fractionation and gtpase-linked immunosorbent assay||Journal of Visualized Experiments||2018||ISSN 1940-087X|
|Itano, Seiji et al.||Colchicine attenuates renal fibrosis in a murine unilateral ureteral obstruction model||Molecular Medicine Reports||2017||ISSN 1791-3004|
|Kim, Jongshin et al.||YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation||Journal of Clinical Investigation||2017||ISSN 1558-8238|
|Ruggiero, Carmen et al.||Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion||Science Signaling||2017||ISSN 1937-9145|
|Paldy, Eszter et al.||Semaphorin 4C Plexin-B2 signaling in peripheral sensory neurons is pronociceptive in a model of inflammatory pain||Nature Communications||2017||ISSN 2041-1723|
|López-Contreras, Luilli et al.||Structural and functional characterization of the divergent Entamoeba Src using Src inhibitor-1||Parasites and Vectors||2017||ISSN 1756-3305|
|Park, Yong Hwan et al.||Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS||Nature Immunology||2016||ISSN 1529-2916|
|Herrera-Martínez, Mayra et al.||Antiamoebic activity of Adenophyllum aurantium (L.) strother and its effect on the actin cytoskeleton of Entamoeba histolytica||Frontiers in Pharmacology||2016||ISSN 1663-9812|
|Nour-Eldine, Wared et al.||Adiponectin attenuates angiotensin II-induced vascular smooth muscle cell remodeling through nitric oxide and the RhoA/ROCK pathway||Frontiers in Pharmacology||2016||ISSN 1663-9812|
|Yan, Yi et al.||Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5||Nature Communications||2015||ISSN 2041-1723|
|Ajima, Rieko et al.||DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assembly||Developmental Biology||2015||ISSN 1095-564X|
|Lasgorceix, M. et al.||In vitro and in vivo evaluation of silicated hydroxyapatite and impact of insulin adsorption||Journal of Materials Science: Materials in Medicine||2014||ISSN 1573-4838|
|Nakajima, Chikako et al.||The lipoprotein receptor LRP1 modulates sphingosine-1-phosphate signaling and is essential for vascular development||Development (Cambridge)||2014||ISSN 1477-9129|
|Moniz, Sónia et al.||Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasiveness||Human molecular genetics||2013||ISSN 1460--2083|
|Herrera-Martínez, M. et al.||Actin, RhoA, and Rab11 participation during encystment in entamoeba invadens||BioMed Research International||2013||ISSN 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 Rabbit||Translational Stroke Research||2013||ISSN 1868-4483|
|Bai, Xue et al.||The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertension||Nature Communications||2013||ISSN 2041-1723|
|Veeramah, Krishna R. et al.||Exome sequencing reveals new causal mutations in children with epileptic encephalopathies||Epilepsia||2013||ISSN 0013-9580|
|Bray, Kristi et al.||Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration||Breast Cancer Research||2013||ISSN 1465-5411|
|Wang, Jiping et al.||RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response||Cardiovascular Diabetology||2012||ISSN 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 pathway||European journal of pharmacology||2012||ISSN 1879--0712|
|Zuo, Yufeng et al.||Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells||Journal of cellular physiology||2012||ISSN 1097--4652|
|Doherty, Jason T. et al.||Skeletal muscle differentiation and fusion are regulated by the BAR-containing Rho-GTPase-activating Protein (Rho-GAP), GRAF||Journal of Biological Chemistry||2011||ISSN 0021-9258|
|Alvarez, S. M. et al.||Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels||British journal of pharmacology||2010||ISSN 1476--5381|
|Heckman-Stoddard, Brandy M. et al.||Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression||Breast Cancer Research : BCR||2009||ISSN 1465-5411|
|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|
|Chastre, Eric et al.||TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness||The FASEB Journal||2009||ISSN 1530--6860|
|Iesato, Ken et al.||Tiotropium bromide attenuates respiratory syncytial virus replication in epithelial cells||Respiration; international review of thoracic diseases||2008||ISSN 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 Formation||Molecular Biology of the Cell||2008||ISSN 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 formation||Molecular Biology of the Cell||2008||ISSN 1059-1524|
|Moniz, Sónia et al.||WNK2 modulates MEK1 activity through the Rho GTPase pathway||Cellular signalling||2008||ISSN 0898--6568|
|Scott, Glynis A. et al.||Lysophosphatidylcholine mediates melanocyte dendricity through PKCzeta activation||The Journal of investigative dermatology||2007||ISSN 1523--1747|
|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|
|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 factor||BMC Biochemistry||2006||ISSN 1471-2091|
|Woods, Anita et al.||RhoA/ROCK Signaling Regulates Chondrogenesis in a Context-dependent Manner *||Journal of Biological Chemistry||2006||ISSN 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.
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).
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.
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.
Any setting that specifies 96 well flat, white will be sufficient.
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 email@example.com.