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).