GEF Exchange Assay

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 (mant-GTP) into GTPases. The uptake can be measured due to the spectroscopic difference between free and GTPase-bound mant-GTP. As mant-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 mant-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)

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

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.

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&rsquo, s GEF activity can be measured using Cytoskeleton&rsquo, 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 µ, M.  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&rsquo, 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 infohoelzel.de