Product Uses Include
The G-LISA series of Small G-Protein Activation Assays are ELISA based assays with which you can measure the GTP form of small G-proteins from lysates of cells or tissues and all 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. The Rac1,2,3 G-LISA™ Activation Assay measures the entire level of GTP-loaded Rac1,2 and 3 protein in cell lysates, this is in contrast to Cat.# BK126 which measures only Rac1 activation levels. The level of activation is measured with absorbance at 490nm. For a kit to measure RhoA activation please check webpage BK124.
The Rac G-LISA Activation Assay is very sensitive and has excellent accuracy for duplicate samples. See G-LISA™ FAQs tab on our G-LISA™ page for more details.
The kit contains sufficient reagents to perform 96 Rac activation assays. Since the Rac-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 Rac activating compound EGF and Rac activation was measured with the G-LISA method (Fig 1 and 2).
Figure 1. Rac activation by EGF measured by G-LISA™ kit BK125. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with EGF (Cal; 10ng/ml for 3 min) or buffer only (SS). 10 µg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm.
Figure 2. Rac activation by EGF measured by G-LISA™. Swiss 3T3 cells were serum starved (SS) for 24 h and treated with EGF (10 ng/ml for 2 min). 20, 10, 5, 2.5, 1.25 µg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm. 500 µg of the same lysates were subjected to the traditional PAK pull-down assay (shown in inset, Cat.# BK035).
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)
|Talwar, Shefali et al.||Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and Rho||Cell Reports||2021||ISSN 2211-1247|
|Lagresle-Peyrou, Chantal et al.||A gain-of-function RAC2 mutation is associated with bone marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency||Haematologica||2021||ISSN 1592-8721|
|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|
|Cash, Jennifer N. et al.||Discovery of small molecules that target the phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) PIP3-binding site and inhibit p-Rex1-dependent functions in neutrophils||Molecular Pharmacology||2020||ISSN 1521-0111|
|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|
|Tsitsilashvili, Elene et al.||Myelin basic protein charge isomers change macrophage polarization||Journal of inflammation research||2019||ISSN 1178--7031|
|Roman-Garcia, Sara et al.||Distinct roles for Bruton's Tyrosine Kinase in B cell immune synapse formation||Frontiers in Immunology||2018||ISSN 1664-3224|
|Hu, Shuling et al.||Mesenchymal Stem Cell Microvesicles Restore Protein Permeability Across Primary Cultures of Injured Human Lung Microvascular Endothelial Cells||Stem cells translational medicine||2018||ISSN 2157--6564|
|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|
|Güntert, Tanja et al.||Temporal Rac1 - HIF-1 crosstalk modulates hypoxic survival of aged neurons||Brain Research||2016||ISSN 1872-6240|
|Chen, Lihua et al.||Smooth muscle-alpha actin inhibits vascular smooth muscle cell proliferation and migration by inhibiting rac1 activity||PLoS ONE||2016||ISSN 1932-6203|
|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|
|Kalia, Manjula et al.||Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism||Journal of Virology||2013||ISSN 0022--538X|
|Teplova, Irina et al.||ATG proteins mediate efferocytosis and suppress inflammation in mammary involution||Autophagy||2013||ISSN 1554-8635|
|Barrio, Laura et al.||TLR4 Signaling Shapes B Cell Dynamics via MyD88-Dependent Pathways and Rac GTPases||The Journal of Immunology||2013||ISSN 0022--1767|
|Ninkovi, Jana et al.||Morphine decreases bacterial phagocytosis by inhibiting actin polymerization through cAMP-, Rac-1-, and p38 MAPK-dependent mechanisms||American Journal of Pathology||2012||ISSN 0002-9440|
|Dhaliwal, Anandika et al.||Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases||2012||PMID 22509380|
|Ridgway, Lon D. et al.||Heparanase-induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells||Molecular Cancer Research||2012||ISSN 1541-7786|
|Halpert, Michal et al.||Rac-dependent doubling of HeLa cell area and impairment of cell migration and cell cycle by compounds from Iris germanica||Protoplasma||2011||ISSN 0033-183X|
|May, James M.||The SLC23 family of ascorbate transporters: ensuring that you get and keep your daily dose of vitamin C||British Journal of Pharmacology||2011||ISSN 0007-1188|
|Vives, Virginie et al.||The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts||2011||ISSN 1523--4681|
|Johanna, Gutiérrez Vargas et al.||Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemia||Neurochemistry international||2010||ISSN 1872--9754|
|Fang, Xiaohui et al.||Allogeneic human mesenchymal stem cells restore epithelial protein permeability in cultured human alveolar type II cells by secretion of angiopoietin-1||Journal of Biological Chemistry||2010||ISSN 0021-9258|
|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|
|Zhu, Jianxin et al.||P21-Activated Kinases Regulate Actin Remodeling in Glomerular Podocytes||American Journal of Physiology - Renal Physiology||2010||ISSN 0363-6127|
|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|
|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|
|Chastre, Eric et al.||TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness||The FASEB Journal||2009||ISSN 1530--6860|
|Baumer, Y. et al.||Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown||Journal of Cellular Physiology||2009||ISSN 1097--4652|
|Abu-Elneel, Kawther et al.||No Title||2008||PMID 18809680|
|Mercer, Jason et al.||Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells||Science (New York, N.Y.)||2008||ISSN 1095--9203|
|Pontow, Suzanne et al.||Antiviral Activity of a Rac GEF inhibitor Characterized with a Sensitive HIV/SIV Fusion Assay||Virology||2007||ISSN 0042-6822|
Question 1: Can I detect isoforms other than RhoA, Rac1,2,3 or RalA with these G-LISA activation assays?
Answer 1: Yes, the RhoA G-LISA (Cat. # BK124), Rac1,2,3 G-LISA (Cat. # BK125) and RalA G-LISA (Cat. # BK129) can be used to detect RhoB or RhoC, Rac 2 or Rac3 or RalB, respectively. To specifically detect Rac1, please see our Rac1 G-LISA activation assay (Cat. # BK128). 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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