RhoGEF exchange assay (BK100)

- Overview
  - 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 consist of more than 150 members, which based on their sequence homology, are divided into several subfamilies such as Rho, Ras, Ran, Rab, Arf and Rem/Rad families. This group of small GTPases serve as binary switches cycling between GDP-bound inactive and GTP-bound active states . The regulatory proteins for this switch include guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). GEFs catalyze the exchange of GDP for GTP to generate the active state of small GTPases in response to extracellular signals. In order to facilitate the exchange, the GEFs must bind to the GDP-bound GTPases, destabilize the GDP-GTPase complex, and then stabilize a nucleotide-free reaction intermediate. Because of the high intracellular ratio of GTP to GDP, the released GDP is replaced with GTP, leading to release of GEFs from the complex and activation of the GTPase. Many GEF proteins have been identified as oncogenes and are involved in human diseases such as cancer. Interestingly, the expression of GEF protein is tissue- or cell-type specific, providing a therapeutic potential for cancer treatment. Recently developed fluorescence analogs of guanine nucleotides have greatly improved the technical ability to define the real-time exchange reaction of GEFs, including kinetic and thermodynamic properties, thereby eliminating the need for traditional radioactive labeling methods. This fluorescence-based assay takes advantage of the spectroscopic difference between bound and unbound fluorescent analogs to guanine nucleotides and resulting in the ability to monitor nucleotide exchange of small GTPases. Once bound to GTPases, the emission intensity of the fluorophore increases dramatically. 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. Cytoskeleton Inc. has developed a N-MAR-GTP fluorophore-based Rho-GEF assay suitable for both 96-well and 384-well formats. This assay can be applied to multiple research purposes such as characterizing the GEFs and identifying GEF inhibitors in a high-throughput screen format. This kit contains human Cdc42, Rac1 and RhoA proteins and the GEF domain of Dbs as a positive control GEF for Cdc42 and RhoA. Dbs shows extremely low GEF activity for Rac1. Interestingly, it was reported that human Dbs can activate Rac1 in a FRET-based assay.
    
    References
    1) Shielge, J. M., et al. Trend Cell Biol., 2000, 10, 147-54.
    2) Whitehead, I. P., et al. Biochim. Biophys. Acta, 1997, 1332, F1-23.
    3) Zheng, Y. Trend Biochem Sci.2002. 26, 724-32.
    4) Cheng, L., et al. Mol. Cell. Biol. 2002. 22, 6895-6905.
    5) Rossman, K. L., et al. Embo J. 2002. 21, 1315-1326.
    6) McEwen, D.P., et al. Anal. Biochem. 2001. 291, 109-117.
    7) Whitehead, I. P., et al. Mol. Cell. Biol. 1999. 19, 7759-7770.
    8) Itoh, R. E., et al. Mol. Cell. Biol. 2002. 22, 6582-6591.
    9) Manor, D. Methods Enzymol. 2000. 325, 139-149.
    
    Please contact us at for specific academic pricing.
- Properties
  - Categories
    
    Small GTPases
    
    * For Research Use Only
- Reference
  - Bogucka-Janczi, Katarzyna et al. ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization. eLife. 2023
    Xu, Yangfan et al. A small Rho GTPase OsRacB is required for pollen germination in rice. Development, Growth & Differentiation. 2022
    Xu, Yangfan et al. A small Rho GTPase OsRacB is required for pollen germination in rice. Development Growth and Differentiation. 2022
    Bharadwaj, Ravi et al. An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1. PLoS Pathogens. 2021
    Giubilaro, Jenna et al. Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen. Nature Communications. 2021
    Eshraghi, Mehdi et al. RasGRP1 is a causal factor in the development of l-DOPA-induced dyskinesia in Parkinson's disease. Science advances. 2020
    Limzerwala, Jazeel F. et al. FoxM1 insufficiency hyperactivates Ect2–RhoA–mDia1 signaling to drive cancer. Nature Cancer. 2020
    Miao, Hui et al. Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction. Journal of Cell Biology. 2019
    Singh, Nikhlesh K. et al. P115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine-induced vascular smooth muscle cell migration and proliferation. Journal of Biological Chemistry. 2017
    Rosenberg, Brian J. et al. Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells. Molecular Biology of the Cell. 2017
    Huang, Guorui et al. WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion. Cell Reports. 2017
    Barreira, María et al. The C-terminal SH3 domain contributes to the intramolecular inhibition of Vav family proteins. Science Signaling. 2014
    Schulz, Alexander et al. Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy. Nature Neuroscience. 2013
    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 activation. The Journal of pharmacology and experimental therapeutics. 2011
    Arbeloa, Ana et al. EspM2 is a RhoA guanine nucleotide exchange factor. Cellular Microbiology. 2010
    Guilluy, Christophe et al. The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure. Nature medicine. 2010
    Oliver, A. W. et al. The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42. British Journal of Cancer 2011 104:2. 2010
    Li, Siwei et al. PLC-γ1 and Rac1 Coregulate EGF-Induced Cytoskeleton Remodeling and Cell Migration. Molecular Endocrinology. 2009
    Kim, Su Jin et al. Dipeptidyl peptidase IV inhibition with MK0431 improves islet graft survival in diabetic NOD mice partially via T-cell modulation. Diabetes. 2009
    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 histolytica. Cellular Microbiology. 2009

Datasheet

MSDS

RhoGEF exchange assay (BK100)

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