RhoA / Rac1 / Cdc42 G-LISA Activation Assay Bundle 3 Kits (24 assays per kit)

RhoA / Rac1 / Cdc42 Activation Assay Bundle - 3 x 24 assays


  • The RhoA / Rac1 / Cdc42 G-LISA Activation Assay Bundle is a cost effective choice when your experimental observations suggest that one or more Rho family proteins are activated.
  • Provides 24 assays each for detection of activated RhoA, Rac1, and Cdc42. 
  • Includes an extensive array of reagents (see datasheet for individual kit contents).
  • Most cost effective and precise method to obtain a Rho family activation profile.

The G-LISA activation assays are ELISA-based activation assays with which you can measure RhoA, Rac1, and Cdc42 activity in as little as 10 ug of cell or tissue lysates in less than 3 hours. These assays are very sensitive and have 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

Kit contents
Each kit contains sufficient reagents to perform 24 activation assays. Larger 96-well assays are also available (see related products). Since the GTP affinity wells are supplied as 12 x 8 well strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from one to multiple assays. For a list of components, please view each of the kit manuals below.

  1. RhoA G-LISA Activation Assay Manual (view pdf here)
  2. Rac1 G-LISA Activation Assay Manual (view pdf here)
  3. Cdc42 G-LISA Activation Assay Manual (view pdf here)


Equipment needed

  1. 96-well plate spectrophotometer capable of reading 490 nm wavelength
  2. Orbital microplate shaker capable of at least 200 rpm shaking (400 rpm is optimal)
  3. Multichannel or multidispensing pipettor (optional)


Example results


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. Cdc42 activation by EGF measured by the Cdc42 G-LISA Activation Assay.  Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with EGF (100 ng/ml for 2 min). Cell lysates (8, 17, 35 µg) were subjected to the G-LISA assay. Data was read at 490 nm.  Numbers on top the yellow columns indicate the fold increase in signal caused by EGF activation, you will notice the ratio remains the same with different protein loadings indicating good linearity with different protein loadings. 500 µg of the same lysates were subjected to the traditional PAK pull-down assay (Cat.# BK034) with similar results.


Associated Products:

Total RhoA ELISA (Cat. # BK150)

Anti-RhoA monoclonal antibody (Cat.# ARH03)

Rho Inhibitor I (Cat.# CT04)

Rho Activator II (Cat.# CN03)

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

RhoA G-LISA Activation Assay Manual (Manual / MSDS)
Rac1 G-LISA Activation Assay Manual (Manual / MSDS)
Cdc42 G-LISA Activation Assay Manual (Manual / MSDS)

    G-LISA Activation Assay Technical Guide (Download Here)


    AuthorTitleJournalYearArticle Link
    Wu, Fei et al.Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflightNature Communications 2024 15:12024ISSN 2041--1723
    Lee, Choong Jae et al.The dysadherin/FAK axis promotes individual cell migration in colon cancerInternational Journal of Biological Sciences2024ISSN 1449-2288
    Sucre, Jennifer M.S. et al.Alveolar repair following LPS-induced injury requires cell-ECM interactionsJCI Insight2023ISSN 2379-3708
    Zhu, Lingxin et al.Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorptionJournal of Cell Biology2023ISSN 1540-8140
    Nataraj, Nishanth Belugali et al.Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factorsCell Reports2022ISSN 2211--1247
    Hassin, Ori et al.Different hotspot p53 mutants exert distinct phenotypes and predict outcome of colorectal cancer patientsNature Communications 2022 13:12022ISSN 2041--1723
    Nataraj, Nishanth Belugali et al.Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factorsCell Reports2022ISSN 2211-1247
    Walter, Lisa Marie et al.Profilin2 regulates actin rod assembly in neuronal cellsScientific Reports2021ISSN 2045-2322
    Rogg, Manuel et al.SRGAP1 controls small rho GTPases to regulate podocyte foot process maintenanceJournal of the American Society of Nephrology2021ISSN 1533-3450
    Ghosh, Soma et al.PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFRCell Reports2021ISSN 2211-1247
    Rodríguez-Fdez, Sonia et al.Lysine Acetylation Reshapes the Downstream Signaling Landscape of Vav1 in LymphocytesCells2020ISSN 2073-4409
    Hung, Pei Shan et al.The inhibition of Wnt restrain KRASG12V-driven metastasis in non-small-cell lung cancerCancers2020ISSN 2072-6694
    Lorenzo-Martín, L. Francisco et al.VAV2 signaling promotes regenerative proliferation in both cutaneous and head and neck squamous cell carcinomaNature Communications2020ISSN 2041-1723
    Lee, Mi Nam et al.The primary cilium directs osteopontin-induced migration of mesenchymal stem cells by regulating CD44 signaling and Cdc42 activationStem Cell Research2020ISSN 1876-7753
    Lorenzo-Martín, L. Francisco et al.Vav2 pharmaco-mimetic mice reveal the therapeutic value and caveats of the catalytic inactivation of a Rho exchange factorOncogene2020ISSN 1476-5594
    Atala, AnthonyRe: MicroRNA-141 Suppresses Prostate Cancer Stem Cells and Metastasis by Targeting a Cohort of Pro-Metastasis GenesThe Journal of urology2017ISSN 1527-3792
    Cerikan, Berati et al.Cell-Intrinsic Adaptation Arising from Chronic Ablation of a Key Rho GTPase RegulatorDevelopmental Cell2016ISSN 1878-1551
    Sarowar, Tasnuva et al.Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2Molecular Brain2016ISSN 1756-6606

    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.


    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.