Vav1 GEF protein (human recomb. DHPHC1 domain Y174D mutant, 6xHis tagged)

On Arrival: 4°C
2(not supplied).

Vav1 protein is a guanine exchange factor with selectivity for Rac1, which mediates Rac1 activation under a variety of conditions and has been associated with multiple diseases for example autoimmune deficiency and esophageal squamous cell carcinoma (reviewed in 1, 2, 3).

Study inhibitors of Vav1 GTP/GDP exchange activity
Identification of Vav1 DHPHC1 domain binding proteins
Study of Vav1 GEF activity with different GTPases

Material

The DHPHC1 domain of human Vav1 protein has been produced in a bacterial expression system. It contains a mutated amino-acid that mimics tyrosine phosphorylation which is required for interaction with Rac1 (Y174D mutant). It is also 6xHis tagged at its amino terminus for purification purposes. The accession number is AAH13361. The molecular weight of GE05 is approximately 41 kDa. The Vav1 protein is supplied as a white lyophilized powder. Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gradient gel. Vav1 DHPHC1 protein was determined to be approximately 80% pure (see Figure 1).

1. Place Vav1 vial on ice and dilute to 0.30 ug/ul (8 uM) with ice cold Exchange Buffer.
2. Dilute Rac protein to 1.25 ug/ul (50 uM) with ice cold Exchange Buffer.
3. Add the following components together in a fresh 15 ml Falcon tube and mix well by pipetting or gentle vortex:
Component ul per well
Exchange Buffer 75
50 uM?Rac 5
8 uM Vav1 10
Note: For a total mixture volume, multiply the volume of reagents per well by the number of wells in the experiment, plus add 20% volume for pipetting losses.
4. Incubate for 20 min at room temperature (RT).
5. Lock in the nucleotide by adding 10 ul (per well) of 50 mM MgCl₂.
6. Set up the fluorimeter with Excitation wavelength at 485 nm +/-15 nm and emission wavelength at 525 nm +/- 15 nm at RT.
7. Aliquot the pre-loaded mixture to the assigned wells and place the plate in the fluorimeter.
8. After 5 cycles (150 seconds), place the program on Hold or Pause, and remove the plate.
9. Pipette 10 ul of a) 5 mM GTP solution, b) a small compound, c) a test protein, d) 4 mM EDTA (+ve exchange control) or e) Dilution Buffer (negative control) in respective wells and immediately pipette up and down twice and resume reading for 20 minutes.
10. Save the readings after the kinetic protocols are finished. The exchange rate can be calculated by reducing the data to max slope (using 12 pts) or Vmax with the software that accompanies the plate reader. The exchange curve can be achieved by export to Microsoft Excel.

Rac1 protein (Cat. # RC01) (2.5 uM) was pre-loaded with Bodipy-FL-GDP using EDTA for exchange. The nucleotide was locked in place with excess Mg2+. Vav1 at different concentrations as shown or Dilution Buffer (purple) was pipetted into wells of a black 384-well low volume plate. At time zero, 500 uM GTP was pipetted in to the wells and the reactions were monitored for 20 min by reading every 30 sec.