RhoGAP assay (BK105)

RhoGAP assay (BK105)

Product Uses Include

  • Determination of the activity and GTPase specificity of uncharacterized GAPs.
  • Biochemical characterization of small GTPases and their associated GAPs.
  • Examination of the regulation of GAP activity by different cofactors or protein domains.
  • Screen the mutant protein of either GAPs or GTPases for activity and substrate specificity.
  • Identification of GAP inhibitors in HTS (high throughput screen) format. Please inquire for significant discounts on large quantities of any reagents in this kit.

The Ras superfamily of small GTPases (such as Ras, Rho, Rab, Arf and Ran proteins) serve as binary switches cycling between a GDP-bound “OFF state” and a GTP-bound “ON state”, which mediates signals downstream. In cells, the cycling between the two states is mainly controlled by two types of regulatory proteins: the activating Guanine nucleotide Exchange Factors (GEFs) and the inactivating GTPase Activating Proteins (GAPs).

Small G-proteins of the Ras superfamily typically have an inrinsic GTPase activity, meaning that after they get activated (GTP-bound) by GEFs, they eventually “turn themselves off”. The intrinsic GTPase rate, however, is normally very slow, so the “automatic” turning off process can take up to hours.

Because of the slow intrinsic GTPase activity in most Ras superfamily GTPases, their GAPs are important signaling molecules that function to terminate their signaling. GAPs function by binding to the active, GTP-bound, form of their target G-proteins and greatly stimulating the G-protein’s intrinsic GTPase activity, thereby quickly turning off the G-protein.

This GAP assay kit measures the amount of inorganic phosphate (Pi) that is produced as a result of G-protein dependent hydrolysis of GTP to GDP + Pi. The kit contains all the reagents needed for the assay including GTPases, a positive control RhoGAP, carefully optimized buffers and phosphate detection reagent. The assay can be performed in either 96-well plates or in 384-well plates. There is enough reagents for 80 assays (20 for each provided GTPase) in the 96-well format and 160 assays (40 for each provided GTPase) in the 384-well format

While the kit comes with Cdc42, Rac1, RhoA and Ras proteins, it can also be used for any other Ras superfamily GTPase. See our G-protein product family web page for other GTPases available from Cytoskeleton.

For a kit to measure GEF activity, see Cat. # BK100.

Kit contents
THe kit contains sufficient materials for 80-160 assays. The following components are included:

  1. Reaction Buffer
  2. His-tagged Cdc42 protein (Cat. # CD01)
  3. His-tagged Rac1 protein(Cat. # RC01)
  4. His-tagged RhoA protein (Cat. # RH01)
  5. His-tagged Ras protein (Cat. # RS01)
  6. GST-tagged p50RhoGAP, catalytic domain (Cat. # GAS01)
  7. GTP stock (Cat. # BST06).
  8. CytoPhos™ reagent.
  9. 96-well clear half area flat bottom plate
  10. Detailed instructions and troubleshooting manual.

Equipment needed

  1. 96-well or 384-well plate spectrophotometer capable of measuring absorbance at 650 nm wavelength

Example results
The GTPase activity of RhoA with and without the addition of the catalytic domain of p50RhoGAP was tested (Fig 1).


Figure 1. RhoGAP activity measured as GTP hydrolysis by RhoA protein.  Each reaction contained reaction buffer + GTP with the addition of RhoA alone (RhoA), RhoGAP alone (GAP), or RhoA + RhoGAP (RhoA + GAP).  Reactions were incubated at 37°C for 20 min. Phosphate generated by hydrolysis of GTP was measured by the addition of CytoPhos™ reagent and reading of absorbance at 650 nm.

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

AuthorTitleJournalYearArticle Link
Lombardo, Andrew T et al.ARHGAP18-ezrin functions as an autoregulatory module for RhoA in the assembly of distinct actin-based structureseLife2024ISSN 2050--084X
Lippincott, Margaret F. et al.The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assayGenetics in Medicine2022
Li, Ching Fei et al.Snail-induced claudin-11 prompts collective migration for tumour progressionNature Cell Biology2019ISSN 1476-4679
Lee, Suho et al.Pyk2 signaling through graf1 and rhoA GTPase is required for amyloid-β oligomer-triggered synapse lossJournal of Neuroscience2019ISSN 1529-2401
Diring, Jessica et al.RPEL-family rhoGAPs link Rac/Cdc42 GTP loading to G-actin availabilityNature Cell Biology2019ISSN 1476-4679
Nüchel, Julian et al.TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulatorsAutophagy2018ISSN 1554-8635
Palsuledesai, Charuta C. et al.Activation of Rho Family GTPases by Small MoleculesACS Chemical Biology2018ISSN 1554-8937
Shin, Yoonjae et al.RASAL3 preferentially stimulates GTP hydrolysisof the rho family small GTPase rac2Biomedical Reports2018ISSN 2049-9442
Lin, Ying Hung et al.RAB10 interacts with the male germ cell-specific GTPase-activating protein during mammalian spermiogenesisInternational Journal of Molecular Sciences2017ISSN 1422-0067
Sarowar, Tasnuva et al.Object phobia and altered RhoA signaling in amygdala of mice lacking RICH2Frontiers in Molecular Neuroscience2017ISSN 1662-5099
Hernandez-Flores, Araceli et al.A new nucleocytoplasmic RhoGAP protein contributes to control the pathogenicity of Entamoeba histolytica by regulating EhRacC and EhRacD activityCellular Microbiology2016ISSN 1462-5822
Bendris, Nawal et al.SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPasesMolecular Biology of the Cell2016ISSN 1939-4586
Zanin, Esther et al.A conserved RhoGAP limits M phase contractility and coordinates with microtubule asters to confine RhoA during CytokinesisDevelopmental Cell2013ISSN 1534-5807
Bai, Xue et al.The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertensionNature Communications2013ISSN 2041-1723
de Kreuk, Bart Jan et al.The Human Minor Histocompatibility Antigen1 Is a RhoGAPPLoS ONE2013ISSN 1932-6203
Xu, Jie et al.RhoGAPs Attenuate Cell Proliferation by Direct Interaction with p53 Tetramerization DomainCell Reports2013ISSN 2211-1247
Taniuchi, Keisuke et al.BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1Neoplasia2012ISSN 1476--5586
Gaus, Kristin et al.Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissueInfection and Immunity2011ISSN 0019-9567
Songsungthong, Warangkhana et al.ROS-inhibitory activity of YopE is required for full virulence of Yersinia in miceCellular Microbiology2010ISSN 1462-5814
Zhang, Ning et al.A novel testis-specific GTPase serves as a link to proteasome biogenesis: Functional characterization of RhoS/RSA-14-44 in spermatogenesisMolecular Biology of the Cell2010ISSN 1059-1524
Köhli, Michael et al.The fuction of two closely related Rho proteins is determined by an atypical switch I regionJournal of Cell Science2008ISSN 0021-9533
Vogt, D. L. et al.ARHGAP4 is a novel RhoGAP that mediates inhibition of cell motility and axon outgrowthMolecular and Cellular Neuroscience2007ISSN 1044-7431
Rothenfluh, Adrian et al.Distinct Behavioral Responses to Ethanol Are Regulated by Alternate RhoGAP18B IsoformsCell2006ISSN 0092-8674


Question 1: How should I prepare my protein for testing in the GAP assay?

Answer 1:  All protein samples and buffers must be free of phosphate prior to beginning the RhoGAP  assay biochem kit (Cat. # BK105).  If your protein is in PBS buffer then it must be dialyzed twice in 1000 volumes of 50 mM PIPES pH 7.0 or 50 mM Tris pH 7.5 buffer to reduce the phosphate content to non-detectable levels.  For the assay itself, prepare an 8X concentration of protein or compound in 20 mM Hepes buffer pH 7.4 or similar or for compounds with low solubility in water, use Milli-Q water and 10% DMSO.  We recommend a final concentration of 30 μM-100 μM for compound screening, or 10 to 1000 nM for proteins.  A titration of your compound/protein is recommended when performing the GAP assay.


Question 2: Can I test cell or tissue lysates with the RhoGAP assay kit to screen for GAP proteins?

Answer 2: No, we do not recommend using lysates from cells or tissue samples with the RhoGAP assay kit (Cat. # BK105).  In lysates there are too many complicating factors for a sensitive and accurate measurement of GAP activity.  Instead, we recommend using a purified compound or protein that has been isolated with immunoprecipitation or purified by His-tagged chromatography. 


Question 3: Does the RhoGAP protein work with Ras GTPase?

Answer 3: The Ras GTPase is not a good substrate for the p50 RhoGAP protein that is included with the RhoGAP assay kit (Cat. # BK105).  We provide the Ras protein as a substrate to screen any potential Ras GAPs that you may wish to study.  Please see these papers for characterization of RhoGAP and its lack of effect on Ras.

Garrett, M. D., Self, A. J., van Oers, C., and Hall, A . (1989) J. Biol. Chem. v 264, pp. 10-13.

Garrett, M. D., Major, G. N., Totty, N., and Hall, A. (1991) Biochem. J. v 276, pp.  833-336.

Lancaster, C. A., Taylor-Harris, P. M., Self, A. J., Brill, S., van Erp, H.E., and Hall, A. (1994) J. Biol Chem. v 269, pp. 1137-1142.



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