We currently offer two fluorescent assay formats that are suitable for measuring the activity of both Ras and Rho family GEFs. These assays measure different steps in the GEF reaction (Fig. 1), which allows them to be leveraged in unique ways. Our original assay offering utilizes mant-GTP to monitor the binding of GTP to a small G-protein in the exchange reaction, and the format of this assay allows for an inhibitor/activator (e.g. small molecule, peptide, protein, etc.) to be pre-incubated with the small G-protein prior to the addition of the GEF. The mant-GTP exchange assay is the foundation of our Rho GEF Exchange Assay Kit (Cat# BK100), which includes hDbs (RhoGEF), RhoA, Rac1, and Cdc42 proteins.
Our latest GEF assay offering monitors the release of GDP during the exchange reaction and utilizes Bodipy-FL-GDP that is pre-bound to the small G-protein of interest. In this assay format, it is possible to pre-incubate an inhibitor/activator with a GEF of interest prior to the addition of the small G-protein:Bodipy-FL-GDP complex. Thus, depending on the nature of your inhibitor design you have the option of a pre-incubation step with either the GEF or small G-protein. Both assay formats work well with our existing (i.e. hDbs) and new Rho and Ras family GEFs (i.e. Vav1, Vav2, Tiam1, and RasGRF1).
Figure 1. The basic elements of the mant-GTP exchange assay that monitors GTP binding to a small G-protein in the exchange reaction (A). Representative data using Sos1 and K-Ras4B in the mant-GTP exchange assay (B). Elements of the Bodipy-FL-GDP exchange assay that monitors GDP release during the exchange reaction (C). Representative data using Sos1 and K-Ras4B in the Bodipy-FL-GDP exchange assay (D).
Currently available GTP Exchange Factor Screens
| Target GEF protein | Description of protein | Compatible Small-G-proteins | Screen Module |
| Dbs | Dbs DHPH domain (aa623-967, human, 6xHis-tagged) | RhoA, RhoB, RhoC, Cdc42 | CDS100 |
| LARG | GEF Domain, human, 6xHis tagged | Rac, Rho | CDS101 |
| RasGRF | RasGRF1 Cdc25 domain (aa1038-1270, human, MBP fusion) | N-Ras, K-Ras, H-Ras, R-Ras | CDS102 |
| RapGEF5 | Rap1 exchange factor, exchange domain, human | Rap1A or 1B | CDS117 |
| SOS1 | Sos1 REM-Cdc25 domain (aa564-1049, human, 6xHis-tagged) | N-Ras, K-Ras, H-Ras | CDS103 |
| SOS2 | Sos2 REM-Cdc25 domain (aa564-1049, human, 6xHis-tagged) | K-Ras4B, N-Ras, H-Ras | CDS118 |
| Tiam1 | Tiam1 DHPH domain (aa1040-1406, human, MBP fusion) | Rac1 | CDS104 |
| Vav1 | Vav1 DHPHC1 domain (aa168-522, human, 6xHis-tagged) with the additional Y174D mutation to mimic Src kinase activation. | Rac1 | CDS111 |
| Vav2 | Vav2 DH domain (aa178-384, human, 6xHis-tagged) | | CDS112 |
GEF Kits and Proteins
Catalog number
Dbs protein GEF protein (Exchange domain) GE01
SOS1 GEF protein (Exchange domain, aa 564-1049) protein CS-GE02
RasGRF1 GEF Protein (Cdc25 Exchange Domain, aa 1038-1270) CS-GE03
Tiam1 GEF Protein (DHPH Exchange Domain, aa 1040-1406) CS-GE04
Vav1 GEF Protein (DHPHC1 Exchange Domain, Y174D mutant, aa 168-522) CS-GE05
Vav2 GEF Protein (DH Exchange Domain, aa 189-374) CS-GE06
ARNO GEF Protein (Exchange domain) CS-GE07
SOS2 GEF Protein (Exchange domain) CS-GE08
RapGEF5 Exchange domain CS-GE09
RhoGEF Exchange Assay BK100
Purified Small G-Proteins and Mutants
H-Ras His-tagged full length human protein RS01
K-Ras4B His-tagged full length human protein CS-RS03
K-Ras4B G12V Mutant His-tagged full length human protein CS-RS04
K-Ras4B G13D Mutant His-tagged full length human protein CS-RS06
K-Ras4B G13S Mutant His-tagged full length human protein CS-RS07
K-Ras4B K128A Mutant His-tagged full length human protein CS-RS08
K-Ras4B Q61P Mutant His-tagged full length human protein CS-RS09
K-Ras4B R135A Mutant His-tagged full length human protein CS-RS10
K-Ras4B G12D+I36N Mutant His-tagged full length human protein, drug binding site tool CS-RS11
K-Ras4B G12D+D38A Mutant His-tagged full length human protein, drug binding site tool CS-RS12
K-Ras4B G12D Mutant His-tagged full length human protein CS-RS13
K-Ras4B G12C Mutant His-tagged full length human protein CS-RS14
N-Ras His-tagged full length human CS-RS02
R-Ras His-tagged full length human CS-RS05
RhoA His-tagged full length human RH01RhoA protein: His tagged: human constitutively active R6301RhoA protein: GST tagged: human wild type RHG01
RhoC His-tagged full length human RH03
Rac1 His-tagged full length human RC01
Rac2 His-tagged full length human RC02Rac1 protein: His tagged: human constitutively active R6101Rac1 protein: GST tagged: human dominant negative R17G01Rac1 protein: GST tagged: human wild type RCG01Cdc42 protein: GST tagged: human wild type CDG01Cdc42 protein: His tagged: human wild type CD01Cdc42 protein: His tagged: human constitutively active C6101
Cdc42 protein: GST tagged: human dominant negative C17G01
Custom GEF Screenings
Dbs Target (DHPH domain, aa 623-967, human, 6xHis-tagged) CDS100RasGRF1 Target (Cdc25 domain, aa 1038-1270, human, MBP fusion) CDS102SOS1 Target (REM-Cdc25 domain, aa 564-1049, human, 6xHis-tagged) CDS103SOS2 Target (REM-Cdc25 domain, human, 6xHis tagged) CDS117
RapGEF5 Target (Exchange domain plus Rap1B protein) CDS118
Tiam1 Target (DHPH domain, aa 1040-1406, human, MBP fusion) CDS104Vav1 Target (DHPHC1 domain, aa 168-522, human, 6xHis-tagged with the additional Y174D mutation to mimic Src kinase activation) CDS111Vav2 Target (DH domain, aa 178-384, human, 6xHis-tagged) CDS112
