RhoC GTPase activation assay.

Abstract

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

• Authors: Michelle Lucey, Heather Unger, Kenneth L van Golen
• Journal: Journal of visualized experiments : JoVE (J Vis Exp) Issue 42 (Aug 22 2010) ISSN: 1940-087X [Electronic] United States
• PMID: 20811327 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Video-Audio Media)

Chemical References

• Apoptosis Regulatory Proteins
• Intracellular Signaling Peptides and Proteins
• RTKN protein, human
• Reagent Kits, Diagnostic
• Recombinant Fusion Proteins
• Glutathione Transferase
• GTP-Binding Proteins
• RHOC protein, human
• rho GTP-Binding Proteins
• rhoC GTP-Binding Protein

Topics

• Apoptosis Regulatory Proteins
• Cell Line
• Enzyme Activation
• GTP-Binding Proteins
• Glutathione Transferase (chemistry, metabolism)
• Humans
• Intracellular Signaling Peptides and Proteins (chemistry, metabolism)
• Reagent Kits, Diagnostic
• Recombinant Fusion Proteins (chemistry, metabolism)
• rho GTP-Binding Proteins (analysis, metabolism)
• rhoC GTP-Binding Protein