Rac1,2,3 G-LISA Activation Assay (Colorimetric format) 96 assays

G-LISA Rac 1,2,3 Activation Assay Biochem Kit (colorimetric format)
$0.00

Product Uses Include

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

Introduction
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.

Kit contents

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:

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

Equipment needed

  1. 96-well plate spectrophotometer capable of reading 490 nm wavelength
  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 cells were stimulated with the Rac activating compound EGF and Rac activation was measured with the G-LISA method (Fig 1 and 2).

BK125_results

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.

bk125_cv

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).

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)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)

Associated Products:
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.   

 

    AuthorTitleJournalYearArticle Link
    Bhosle, Vikrant K. et al.The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureuseLife2023ISSN 2050-084X
    Barbakadze, Tamar et al.3,5,3'-Triiodo-L-Thyronine Regulates Actin Cytoskeleton Dynamic in The Differentiated PC-12 Cells during Hypoxia through An αvβ3 IntegrinCell Journal (Yakhteh)2023ISSN 2228-5814
    Talwar, Shefali et al.Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and RhoCell Reports2021ISSN 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 immunodeficiencyHaematologica2021ISSN 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 ProteinsThe Journal of eukaryotic microbiology2020ISSN 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 neutrophilsMolecular Pharmacology2020ISSN 1521-0111
    Tsitsilashvili, Elene et al.Myelin basic protein charge isomers change macrophage polarizationJournal of inflammation research2019ISSN 1178--7031
    Wu, Xuping et al.Wnt5a induces ROR1 and ROR2 to activate RhoA in esophageal squamous cell carcinoma cellsCancer Management and Research2019ISSN 1179-1322
    Hu, Shuling et al.Mesenchymal Stem Cell Microvesicles Restore Protein Permeability Across Primary Cultures of Injured Human Lung Microvascular Endothelial CellsStem cells translational medicine2018ISSN 2157--6564
    Roman-Garcia, Sara et al.Distinct roles for Bruton's Tyrosine Kinase in B cell immune synapse formationFrontiers in Immunology2018ISSN 1664-3224
    Chen, Lihua et al.Smooth muscle-alpha actin inhibits vascular smooth muscle cell proliferation and migration by inhibiting rac1 activityPLoS ONE2016ISSN 1932-6203
    Güntert, Tanja et al.Temporal Rac1 - HIF-1 crosstalk modulates hypoxic survival of aged neuronsBrain Research2016ISSN 1872-6240
    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
    Ahn, Bum Ju et al.Ninjurin1 enhances the basal motility and transendothelial migration of immune cells by inducing protrusive membrane dynamicsJournal of Biological Chemistry2014ISSN 1083-351X
    Kalia, Manjula et al.Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic MechanismJournal of Virology2013ISSN 0022--538X
    Teplova, Irina et al.ATG proteins mediate efferocytosis and suppress inflammation in mammary involutionAutophagy2013ISSN 1554-8635
    Barrio, Laura et al.TLR4 Signaling Shapes B Cell Dynamics via MyD88-Dependent Pathways and Rac GTPasesThe Journal of Immunology2013ISSN 0022--1767
    Dhaliwal, Anandika et al.Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases2012PMID 22509380
    Ninkovi, Jana et al.Morphine decreases bacterial phagocytosis by inhibiting actin polymerization through cAMP-, Rac-1-, and p38 MAPK-dependent mechanismsAmerican Journal of Pathology2012ISSN 0002-9440
    Ridgway, Lon D. et al.Heparanase-induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cellsMolecular Cancer Research2012ISSN 1541-7786
    Vives, Virginie et al.The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts2011ISSN 1523--4681
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    Johanna, Gutiérrez Vargas et al.Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemiaNeurochemistry international2010ISSN 1872--9754
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    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.

     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.