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Vav2 GEF protein (human recomb. DH domain, 6xHis tagged)

Vav2 GEF protein (human recomb. DH domain, 6xHis tagged)

Item no.	CS-GE06
Manufacturer	Cytoskeleton
Amount	1 x 100 ug
Category	Peptides & Proteins Recombinant Proteins
Type	Proteins
Specific against	other
Purity	Protein purity is determined by scanning densitometry of Coomassie blue stained protein on a 4-20% polyacrylamide gradient gel. The Vav2 protein was determined to be c. 80% pure. (see Figure 1).
ECLASS 10.1	32160409
ECLASS 11.0	32160409
UNSPSC	12352202
Shipping condition	Room temperature
Available
Shipping Temperature	AT
Storage Conditions	On Arrival: 4°C Before reconstitution, briefly centrifuge to collect the product at the bottom of the tube. The protein should be reconstituted to 5 mg/ml with ice cold nanopure water (20 ul water per 100 ug protein).When reconstituted, the protein will be in the following buffer: 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2, 5% (v/v) sucrose and 1% (v/v) dextran. In order to maintain high biological activity of the protein it is strongly recommended that the protein solution be supplemented with DTT to 1 mM final concentration, aliquoted into "experiment sized" amounts, snap frozen in liquid nitrogen and stored at -70C. The protein is stable for six months if stored at -70C. The protein must not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated (< 10% humidity) for one year. Further Vav2 dilutions should be made in 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl₂(not supplied).
Weight (grams)	8
Product Uses	Vav2 protein is a guanine exchange factor with selectivity for Rac1, which mediates Rac1 activation under a variety of conditions and has been associated with gastric tumors as well as cellular volume control and neurite extension (reviewed in 1, 2, 3). Study inhibitors of Vav2 GTP/GDP exchange activity Identification of Vav2 DH domain binding proteins Study of Vav2 GEF activity with different GTPases
Material	The DH domain of human Vav2 protein has been produced in a bacterial expression system. It contains a 6xHis tagged at its amino terminus for purification purposes. The accession number is NM_001134398.1. The molecular weight of GE06 is approximately 25 kDa. The Vav2 DH 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. Vav2 DH protein was determined to be approximately 80% pure (see Figure 1).
Reagents	1. Vav2 DH protein (Cat. # CS-GE06) 2. Rac1, 2 or 3 protein (Cat. # RC01, CS-RC02 or CS-RC03) 3. Exchange buffer 2 (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 2 mM EDTA, 100 ug/ml BSA, and 0.75 uM Bodipy-FL-GDP), note - make fresh. 4. 50 mM MgCl₂in 20 mM Tris-HCl pH 7.5, 50 mM NaCl. 5. 5 mM GTP in 20 mM Tris-HCl pH 7.5, 50 mM NaCl.
Method	1. Place Vav2 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 Vav2 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. Legend: 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⁺. Vav2 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.
Equipment needed	1. Fluorescence spectrometer (?ex=485nm, ?em=525nm, both with< 15nm bandwidth) 2. Corning 96-well half area plates (Cat # 3686) or other plate with low protein binding surface.
Figure 1 Legend	Legend: A 5 ug sample of recombinant Vav2 DH (molecular weight approx. 25 kDa) was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Protein quantitation was determined using the Precision Red Protein Assay Reagent (Cat. # ADV02). ?Mark12 molecular weight markers are from Life Technologies Inc.

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