Product Uses Include
The human H-Ras protein has been produced in a bacterial expression system. The protein is supplied as a lyophilized powder. When it is reconstituted in distilled water to 1 mg/ml, the protein is in the following buffer: 2 mM Tris pH 7.6, 0.5 mM MgCl2, 0.5% sucrose, 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent, Cat. # ADV02.
The recombinant protein is approximately 28 kDa, consisting of the H-Ras protein plus a histidine tag in the amino-terminus.
For other forms of Ras as well as many other purified small G-proteins, see our main small G-protein product page.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. His-H-Ras samples are >80% pure.
Figure 1: His-H-Ras protein purity determination. A 10 µg sample of RS01 (His-H-Ras molecular weight approx. 28 kDa) was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue.
The biological activity of His-H-Ras is determined from its ability to catalyze the exchange of GDP for GTP. EDTA is used to sequester magensium ions from the His-H-Ras protein, thereby stimulating nucleotide exchange activity. The RhoGEF exchange assay Biochem Kit™ (Cat. # BK100) is used to monitor the nucleotide exchange in His-H-Ras.
Figure 2: His-Ras exchange assay using BK100. His-H-Ras protein (1 µM) was mixed with exchange buffer and aliquoted to four wells of a 96-well half area plate. After 5 cycles of reading in a fluorimeter, EDTA was added to 40 mM in the test samples and Milli-Q water to the control samples. Reactions were monitored for 30 min.
|Kovalski, Joanna R. et al.||The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2||Molecular Cell||2019||ISSN 1097-4164|
|Surviladze, Zurab et al.||Identification of a Small GTPase Inhibitor Using a High-Throughput Flow Cytometry Bead-Based Multiplex Assay||SLAS Discovery||2010||ISSN 2472--5552|