Product Uses
Drug binding studies with the compounds that bind in the groove occupied by K128.
Study of K128A K-Ras4B exchange activity with different GEFs
Identification of K128A K-Ras4B exchange factors (GEFs)
Positive control for GEF studies
Biochemical characterization of K128A K-Ras4B protein interactions
Western blot standard
Materials
The K128A (lysine to alanine at amino acid position 128) mutant 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 K128A K-Ras4B is approximately 25 kDa and it is supplied as a white lyophilized powder. Note: The K128A mutation endows the protein with endogenous exchange activity even in the presence of Mg2+ ions (see Figure 2).
Storage
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 μl of Milli-Q water (100 μg 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 at -70°C. The protein is stable for six months if stored at - 70°C. The protein should not be exposed to repeated freezethaw cycles. The lyophilized protein is stable at 4°C desiccated (<10% humidity) for one year.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue-stained protein on a 4-20% polyacrylamide gradient gel. His tagged K128A K-Ras4B protein was determined to be >90% pure. (see Figure 1).
Figure 1. K128A K-Ras4B Protein Purity Determination.
Legend: A 20 μg sample of recombinant K128A 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). SeeBlue molecular weight markers are from Life Technologies Inc.
Biological Activity Assay
The biological activity of K128A K-Ras4B can be determined from the ability of the SOS1 exchange domain (SOS1-ExD) to catalyze the exchange of GDP for GTP onto mutant K-Ras4B. A standard biological assay for monitoring the biological activity of K128A KRas4B is an exchange assay utilizing the 2X Exchange Buffer from the RhoGEF exchange assay kit (Cat.# BK100). The Exchange Buffer contains Mant-GTP as reporter, which can be changed for Bodipy-GDP or -GTP for 490/513nm range and greater sensitivity.