« Back
Home /
K-Ras4B Protein: wild-type (human recombinant with 6xHis-tag)

K-Ras4B Protein: wild-type (human recombinant with 6xHis-tag)

Item no.	CS-RS03
Manufacturer	Cytoskeleton
Amount	1 x 100 ug
Category	Oncology By Cell Type Blastoma Carcinoma Glioma Hematology Leukemia Lymphoma Myeloma Melanoma Sarcoma Peptides & Proteins Recombinant Proteins Cell Biology Adenocarcinoma Cytoskeleton By Organ Lung Cancer Colorectal Cancer small GTPases Ras Subfamily
Type	Proteins
Specific against	Human (Homo sapiens)
Purity	Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gradient gel. His tagged K-Ras4B protein was determined to be >90% 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 the addition of 20 ul of Milli-Q water (100 ug size). When reconstituted, the protein will be in the following buffer: 50 mM Tris pH 7.5, 50 mM NaCl, 0.5 mM MgCl₂ 5% (w/v) sucrose and 1% (w/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 a -70C. The protein is stable for six months if stored at -70C.The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated (< 10% humidity) for one year.
Description	* Limited stock available. If stock is not available, Cytoskeleton will produce a new batch upon request. Minimum order will apply. Inquire for more information.
Weight (grams)	8
Product Uses	Study of K-Ras4B exchange activity with different GEFs. Identification of K-Ras4B exchange factors (GEFs) Positive control for GEF studies. Biochemical characterization of K-Ras4B protein interactions Western blot standard
Material	The wild-type human K-Ras4B protein has been produced in a bacterial expression system. The recombinant protein contains six histidine residues at its amino terminus (His-tag). The molecular weight of 6xHis tagged K-Ras4B is approximately 25 kDa and is supplied as a white lyophilized powder.
Biological Activity	The biological activity of K-Ras4B can be determined from the ability of the SOS1 exchange domain (SOS1-ExD) to catalyze the exchange of GDP for GTP on K-Ras4B. A standard biological assay for monitoring the biological activity of K-Ras4B is an exchange assay utilizing the 2X Exchange Buffer from the Rho-GEF exchange assay kit (Cat.# BK100) and the human SOS1 GEF domain (Cat.# CS-SOS1).
Reagents	1. Recombinant K-Ras4B protein (Cat.# CS-RS03) 2. Recombinant SOS1-ExD protein (Cat.# CS-SOS1) 3. 2x Exchange Buffer (40 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl₂ 0.1 mg/ml BSA, 1.5 uM mant-GTP) 4. Dilution Buffer (20 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl₂ 0.1 mg/ml BSA) Equipment 1. Fluorescence spectrometer (?ex=360nm, ?em=440nm) 2. Corning 96-well half area plates (Cat # 3686) or other plate with low protein binding surface.
Method	1. Dilute SOS1-ExD protein (Cat# CS-SOS1) to 1 uM (0.06 mg/ml) with Dilution Buffer. 2. Dilute K-Ras4B to 50 uM (1.1 mg/ml) with Dilution Buffer. 3. Dissolve lyophilized 2x Exchange Buffer in 5 ml Milli-Q?water and keep in room temperature. 4. Set up the plate reader for kinetic fluorescence measurements (Excitation wavelength at 360 nm and emission wavelength at 440 nm) with readings every 30 seconds for 30 minutes. 5. Add the following components together and mix well by gentle pipetting: Exchange reaction mix / 96 well black plate 2x Exchange Buffer / 50 ?l dH2O / 26 ?l 50 ?M K-Ras4B / 4 ?l 6. Pipette 20 ul of 1 uM SOS1-ExD protein or Dilution Buffer in their respective wells and immediately pipette up and down twice and begin reading the fluorescence. 7. Once the readings are complete and the plate reader file has been saved, the exchange rate can be calculated by reducing the data to Vmax with the software that accompanies the plate reader.
Figure 1 Legend	Legend: A 20 ug sample of recombinant K-Ras4B protein (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.

Note: The presented information and documents (Manual, Product Datasheet, Safety Datasheet and Certificate of Analysis) correspond to our latest update and should serve for orientational purpose only. We do not guarantee the topicality. We would kindly ask you to make a request for specific requirements, if necessary.

All products are intended for research use only (RUO). Not for human, veterinary or therapeutic use.

CS-RS03.pdf

CS-RS03-datasheet.pdf

CS-RS03-safety-datasheet.pdf