RhoGEF exchange assay (BK100)

RhoGEF exchange assay (BK100)
$0.00

Product Uses Include

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

Introduction
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”. 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).

GEFs function by catalyzing the exchange of nucleotide on their target GTPases. The GEF will bind to a nucleotide-bound GTPase, which causes the bound nucleotide to be released, thus resulting in a nucleotide free GEF-GTPase reaction intermediate. This nucleotide free complex will then take up a new nucleotide after which the GEF is released from the GTPase. Because GEFs typically have a higher affinity for GDP-bound GTPases than for the corresponding GTP-bound GTPase and the intracellular ratio of GTP to GDP is about 10:1, GEFs will drive the exchange from GDP-bound GTPases to GTP-bound GTPases

The fluorophore based assay in this kit is suitable for measuring nucleotide exchange on GTPases in either 96-well or 384-well format. The kit measures the uptake of the fluorescent nucleotide analog N-methylanthraniloyl-GTP (N-MAR-GTP) into GTPases. The uptake can be measured due to the spectroscopic difference between free and GTPase-bound N-MAR-GTP. As N-MAR-GTP gets bound in the nucleotide binding pocket of a GTPase, its fluorescence (ex: 360 nm, em: 440 nm) increases dramatically (1, 2). Therefore, the enhancement of N-MAR-GTP fluorescent intensity in the presence of a small GTPase indicates nucleotide uptake (or exchange for already bound nucleotide) by the GTPase.

This kit contains all the buffers and reagents needed for the assay, a 96 and a 384-well plate, as well as human Cdc42, Rac1 and RhoA GTPases and the GEF domain of Dbs as a positive control GEF. Dbs is an effective GEF for Cdc42 and RhoA but shows only very low GEF activity for Rac1 (see Figs 1 & 2). The kit provides enough material for 20 assays for each GTPase in 96-well format or 100 assays for each GTPase in 384-well format.

While the kit comes with Cdc42, Rac1 and RhoA 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 GAP activity, see Cat. # BK105

Kit contents
The kit contains enough materials for 60 assays in 96-well format or 300 assays in 384-well format. The following components are included:

  1. Exchange 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 hDbs protein (Cat. # GE01)
  6. 384-well black half area round bottom plate
  7. 96-well black half area flat bottom plate
  8. Detailed instructions and troubleshooting manual.

Equipment needed

  1. Fluorescence plate reader (96 or 384-well) capable of measuring fluorescence at ex: 360 nm, em: 440 nm

Example results
The exchange activity of Dbs on Cdc42, RhoA and Rac1 was measured in 96-well and 384-well format (Fig 1 & 2)

bk100_f1

Figure 1. Dbs exchange activity for Cdc42, RhoA and Rac1 in 96-well half area plate format

bk100_f2

Figure 2. Dbs exchange activity for Cdc42, RhoA and Rac1 in 384-well plate format

References

  • John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R. S. (1990) Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry 29, 6058-6065.
  • Leonard, D. A., Evans, T., Hart, M., Cerione, R. A., and Manor, D. (1994) Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry 33, 12323-12328.

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
Xu, Yangfan et al.A small Rho GTPase OsRacB is required for pollen germination in riceDevelopment Growth and Differentiation2022ISSN 1440-169X
Bharadwaj, Ravi et al.An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1PLoS Pathogens2021ISSN 1553-7374
Giubilaro, Jenna et al.Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screenNature Communications2021ISSN 2041-1723
Eshraghi, Mehdi et al.RasGRP1 is a causal factor in the development of l-DOPA-induced dyskinesia in Parkinson's diseaseScience advances2020ISSN 2375--2548
Limzerwala, Jazeel F. et al.FoxM1 insufficiency hyperactivates Ect2–RhoA–mDia1 signaling to drive cancerNature Cancer2020ISSN 2662-1347
Miao, Hui et al.Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constrictionJournal of Cell Biology2019ISSN 1540-8140
Singh, Nikhlesh K. et al.P115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine-induced vascular smooth muscle cell migration and proliferationJournal of Biological Chemistry2017ISSN 1083-351X
Rosenberg, Brian J. et al.Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cellsMolecular Biology of the Cell2017ISSN 1939-4586
Huang, Guorui et al.WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial FusionCell Reports2017ISSN 2211-1247
Barreira, María et al.The C-terminal SH3 domain contributes to the intramolecular inhibition of Vav family proteinsScience Signaling2014ISSN 1937-9145
Schulz, Alexander et al.Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathyNature Neuroscience2013ISSN 1097-6256
Moey, Melissa et al.Ginseng (Panax quinquefolius) attenuates leptin-induced cardiac hypertrophy through inhibition of p115Rho guanine nucleotide exchange factor-RhoA/Rho-associated, coiled-coil containing protein kinase-dependent mitogen-activated protein kinase pathway activationThe Journal of pharmacology and experimental therapeutics2011ISSN 1521--0103
Guilluy, Christophe et al.The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressureNature medicine2010ISSN 1546--170X
Oliver, A. W. et al.The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42British Journal of Cancer 2011 104:22010ISSN 1532--1827
Arbeloa, Ana et al.EspM2 is a RhoA guanine nucleotide exchange factorCellular Microbiology2010ISSN 1462-5814
Li, Siwei et al.PLC-γ1 and Rac1 Coregulate EGF-Induced Cytoskeleton Remodeling and Cell MigrationMolecular Endocrinology2009ISSN 0888--8809
Kim, Su Jin et al.Dipeptidyl peptidase IV inhibition with MK0431 improves islet graft survival in diabetic NOD mice partially via T-cell modulationDiabetes2009ISSN 1939--327X
Nakada-Tsukui, Kumiko et al.Phosphatidylinositol-phosphates mediate cytoskeletal reorganization during phagocytosis via a unique modular protein consisting of RhoGEF/DH and FYVE domains in the parasitic protozoon Entamoeba histolyticaCellular Microbiology2009ISSN 1462-5814

 

Question 1: What is the best way to measure GEF activity from a cell lysate?

Answer 1:  First, the GEF protein must be immunoprecipitated from the cell lysate with either an antibody to the protein or to a tag (His, GST, etc.) that has been conjugated to the protein of interest.  After eluting your protein of interest from the beads, the protein’s GEF activity can be measured using Cytoskeleton’s RhoGEF Exchange Assay Biochem Kit (Cat. # BK100).  Cytoskeleton Inc. has developed a mant fluorophore-based GEF assay designed for characterizing GEFs and identifying GEF inhibitors.  This kit contains human Cdc42 (Cat. # CD01), Rac1 (Cat. # RC01) and RhoA (Cat. # RH01) proteins and the GEF domain of Dbs (Cat. # GE01) as a positive control GEF for Cdc42 and RhoA.  The kit also comes with a 384-well and 96-well plate along with exchange buffer that contains the mant-GTP.  Once bound to GTPases, the fluorophore emission intensity of mant-GTP increases approximately 1.1 to 2.0 fold depending on the protein. Therefore, the enhancement of fluorescent intensity in the presence of small GTPases and GEFs will reflect the respective GEF activities of known or unknown proteins.  We recommend titrating the concentration of your GEF protein to optimize its activity.  We suggest a titration range of 0.01-1 µM.  A citation that used our GEF assay kit with an immunoprecipitated protein is Kakiashvili et al., 2009.  GEF-H1 mediates tumor necrosis factor-alpha-induced Rho activation and myosin phosphorylation: role in the regulation of tubular paracellular permeability.  J. Biol. Chem. 284, 11454-11466.

 

Question 2: I want to use the RhoGEF Exchange Assay kit with a 384 well plate.  Is this possible?

Answer 2:  Yes, Cytoskeleton’s mant fluorophore based GEF assay (Cat. # BK100) is suitable for a 384 well high-throughput screen format.  Up to 130 reactions per GTPase can be achieved if a 384-well low volume black round bottom plate (Corning Cat# 3676) is used. This type of 384 well plate is strongly recommended due to the greater signal/noise ratio. Do not use clear plates since that will give you significant background noise.  We provide a 384 well plate and a protocol for this HTS format in the manual that comes with the kit.

 

 

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