Product Uses Include
The human RhoC 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 30 kDa, consisting of the RhoC protein plus a histidine tag in the amino-terminus.
For other forms of Rho proteins 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-RhoC samples are ~90% pure.
Figure 1: His-RhoC protein purity determination. A 10 µg sample of RH01 (His-RhoC molecular weight approx. 30 kDa) was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue.
The biological activity of His-RhoC is determined from its ability to catalyze the exchange of GDP for GTP. EDTA is used to sequester magensium ions from the His-RhoC protein, thereby stimulating nucleotide exchange activity. The RhoGEF exchange assay biochem kit (Cat. # BK100) is used to monitor the exchange ability of His-RhoC.
Figure 2: His-RhoC exchange assay using BK100. His-RhoC 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 to 40 mM or Milli-Q water were added to the wells and the reactions monitored for 30 min.