Product Uses Include
1. Determination of the activity of unknown GEFs.
2. Biochemical characterization of small GTPases and their associated GEFs.
3. Examination of the regulation of GEF activity by different cofactors or protein domains.
4. Screening mutant proteins of either GEFs or GTPases for activity and substrate specificity.
5. Identify of GEF inhibitors in an HTS (high throughput screen) format.
Introduction
Recently developed fluorescence analogs of guanine nucleotides allow scientists to take advantage of fluorescence-based assays to measure the spectroscopic differences between bound and unbound fluorescent analogs to guanine nucleotides and resulting in the ability to monitor nucleotide exchange of small GTPases (1,2). Once bound to GTPases, the emission intensity of the fluorophore increases dramatically (Fig. 1). Therefore, enhancement of fluorescent intensity in the presence of small GTPases and GEFs will reflect the respective GEF activities of known or unknown proteins. While the kit comes with K-Ras and SOS1 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.
Figure legend
KRAS-4B and SOS1 exchange assay: The small GTPase KRAS-4B (Cat# CS-RS03) and SOS1 (Cat# GE02) were resuspended as described in the manual. The reactions were conducted in a 96-well black flat bottom half area plate (Corning Cat# 3686) format (100 µl reaction volumes). Each reaction contains 2 µM KRAS-4B, 20 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl2, and 1.5 µM BODIPY FL GTP with or without 0.8 µM human SOS1(Exchange domain) protein. Reactions were measured in a Spectrofluor M2 (top image) or SpectraMax iD5 fluorimeter bottom image (ex= 485 nm, em= 535nm). Readings were taken at 20°C every 30 seconds for a total reaction time of 30 minutes. Top image indicates the basic 96-well reaction performance. Bottom images indicates 384-well N-MAR-GTP dissociation format with K-Ras inhibitor concentration denoted on the right.
Kit contents
The kit contains enough materials for 80 assays in 96-well format or 400 assays in 384-well format. The following components are included:
- Exchange Buffer
- His-tagged K-Ras protein (Cat. # CS-RS03)
- His-tagged SOS1 protein(Cat. # CS-GE02)
- 384-well black half area round bottom plate
- 96-well black half area flat bottom plate
- Detailed instructions and troubleshooting manual.
Equipment needed
- Fluorescence plate reader (96 or 384-well) capable of measuring fluorescence at ex: 485 nm, em: 515 nm
For a kit to measure GAP activity, see Cat. # BK105
References
- 1) Hong, D.S., et al., N. Engl. J. Med., 383, 1207-1217 (2020)
2) Cheng, L., et al., Mol. Cell. Biol., 22, 6895-6905 (2002)
There are two methods suitable for measuring GTP-exchange activity of Ras family GEFs and Ras proteins. 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).
For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
BK101 is a newly introduced kit in 2024, therefore no citations currently exist. However, the Rho GTP-exchange analogous kit, Cat.# BK100, has a similar format and thus is representing the exchange assay format with these citations:
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 Ras GEF Exchange Assay Biochem Kit (Cat. # BK101). Cytoskeleton Inc. has developed a Bodipy-FL fluorophore-based GEF assay designed for characterizing GEFs and identifying GEF inhibitors. This kit contains human K-Ras (Cat. # RS03), and the GEF domain of SOS1 (Cat. # GE02) as a positive control GEF. The kit also comes with a 384-well and 96-well plate along with exchange buffer that contains the Bodipy-FL-GTP. Once bound to GTPases, the fluorophore emission intensity increases approximately 1.3 to 2.0 fold depending on the GTPase 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 GEF Exchange Assay kit with a 384 well plate. Is this possible?
Answer 2: Yes, Cytoskeleton’s mant fluorophore based GEF assays (Cat. # BK100 and BK101) are 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
