Rhotekin RBD protein GST fusion

Question 1: Can the Rhotekin-RBD protein be used to localize active small G-proteins in cells?

Answer 1: GST-tagged rhotekin-RBD (Cat. #RT01) proteins has been used to identify activated small G-proteins in fixed and permeabilized cells. Examples of this technique are reported in Berdeaux et al., 2004 (Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function. J. Cell Biol. 166, 317-323) and Zhao et al., 2007 (Force activates smooth muscle -actin promoter activity through the Rho signaling pathway. J. Cell Sci. 120, 1801-1809). Cytoskeleton has not verified this technique as a viable and accurate means of localizing activated small G-proteins and because it is not a widely utilized technique, we cannot confirm that this procedure achieves an accurate representation of activated GTPases. Briefly, cells are grown on glass coverslips, treated with control and experimental conditions, fixed, permeabilized and incubated with Rhotekin-RBD proteins. Localization of the bound proteins is accomplished by immunocytochemical detection of the GST tag with an anti-GST antibody that will allow localization of the activated small G-proteins bound by Rhotekin-RBD.

Question 2: Can the rhotekin-RBD protein be used to measure activated RhoB and RhC levels in lysates?

Answer 2: Yes, the rhotekin-RBD protein (Cat. #RT01) can be used to examine activated (GTP-bound) RhoA, RhoB, or RhC in cell or tissue lysates. To detect a specific Rho isoform, a monoclonal antibody that has been tested and had its specificity verified to the target Rho isoform is recommended. In addition, we recommend using a purified version of the protein as a positive control with each western blot experiment.