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Ras-GRF GEF protein (human recomb. CDC25 domain, MBP tagged)

Ras-GRF GEF protein (human recomb. CDC25 domain, MBP tagged)

ArtNr	CS-GE03
Hersteller	Cytoskeleton
Menge	1 x 100 ug
Kategorie	Neuroscience Neural Lineage Markers Molecular Targets for Neuroscience Targets for Rodent Neurosciences Peptides & Proteins Recombinant Proteins Astrocyte Markers
Typ	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 SOS1-ExD protein was determined to be >90% pure. (see Figure 1).
ECLASS 10.1	32160409
ECLASS 11.0	32160409
UNSPSC	12352202
Versandbedingung	Raumtemperatur
Lieferbar
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 Ras-GRF1 dilutions should be made in 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2.(not supplied).
Weight (grams)	8
Product Uses	RasGRF1 is a guanine exchange factor with selectivity for H-, K-, N- and R-Ras, which mediates Ras isoform activation under a variety of conditions and has been associated with multiple diseases (reviewed in 1, 2). Study inhibitors of RasGRF GTP/GDP exchange activity Identification of RasGRF Cdc25 domain binding proteins Study of RasGRF GEF activity with different GTPases
Material	The Cdc25 domain of human RasGRF1 protein has been produced in a bacterial expression system. It contains an MBP fusion at its amino terminus. The accession number is NM_002891.4. The molecular weight of MBP-RasGRF1 is approximately 60 kDa. The RasGRF1 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. MBP-RasGRF1 was determined to be > 85% pure.
Biological Activity	The biological activity of RasGRF1 CDC25 domain can be determined from its ability to catalyze nucleotide exchange on Ras isoforms using the nucleotide exchange assay of Bodipy-GDP for excess GDP or GTP. Ras protein is pre-loaded with Bodipy-FL-GDP by adding excess EDTA e.g. 0.7 mmol EDTA per mmol Mg2+ ions present in the reaction. This sub-stock solution is then used in a dissociation assay format which indicates competition for exchange site with unlabeled nucleotide. The reaction is monitored by fluorescence measurement at 485nm Ex / 535nm Em. Stringent quality control ensures that the exchange rate of Bodipy-GTP or mant-GTP is enhanced at least five fold in the presence of 0.8 uM RasGRF CDC25 domain. Reagents 1. Recombinant Ras protein (Cat.# RS01hH-Ras, RS02hN-Ras, RS03hK-Ras, RS04hK-Ras-G12V, RS05hR-Ras. 2. Recombinant RasGRF Cdc25 protein domain (Cat.# CS-GE03) 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 MgCl2 in 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. Equipment 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.
Method	1. Place RasGRF vial on ice and dilute to 0.40 ug/ul (8 uM) with ice cold Exchange Buffer. 2. Dilute Ras 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?Ras 5 8 uM RasGRF 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 +/-20 nm and emission wavelength at 535 nm +/- 20 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: K-Ras4B protein (Cat. # RS03) (2.5 uM) was pre-loaded with Bodipy-FL-GDP using EDTA for exchange. The nucleotide was locked in place with excess Mg2+. RasGRF 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.
Figure 1 Legend	Legend: A 20 ug sample of recombinant Ras-GRF1 (Cat.# CS-GE03 with molecular weight approx. 60 kDa) was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Protein was determined using the Precision Red Protein Assay Reagent (Cat. # ADV02). ?Mark12 molecular weight markers are from Life Technologies Inc.

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