Rhotekin-RBD protein (binds active Rho proteins)

Rhotekin-RBD protein (binds active Rho proteins)

Product Uses Include

  • Precipitation of active Rho from cell lysates
  • Inhibition of RhoA/B/C activity in vitro
  • Blocking of RhoA/B/C function in vivo by microinjection

The Rho binding domain (RBD) of the human Rhotekin protein has been expressed as a GST-fusion protein in E. coli. This protein binds binds specifically to GTP-bound, and not GDP-bound, RhoA, RhoB and RhoC proteins. The domain can therefore be used to specifically precipitate active, GTP-bound Rho proteins as well as to specifically block the activity of RhoA/B/C in vitro and in vivo.

The GST-Rhotekin-RBD contains residues 7-89 of Rhotekin. This region includes the sequences required for the high affinity interaction with GTP-Rho.

The protein is supplied as 500 µg of lyophilized product and the protein is also available in a bead bound format (Cat. # RT02). This product is used in our RhoA pulldown activation assay Biochem Kit™ (Cat. # BK036)

Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-Rhotekin-RBD protein is >90% pure (see Figure 1).


Figure 1:  GST-Rhotekin-RBD protein purity determination. A 10 µg sample of RT01 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue.

Biological Activity
The Rhotekin-RBD protein specifically recognizes and binds to the active, GTP-bound, form of RhoA, RhoB or RhoC protein. It has a much lower affinity for the inactive, GDP-bound, form of RhoA/B/C. Biological activity of Rhotekin-RBD protein is therefore determined by its selectivity for GTP-RhoA protein.

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Li, Yijian et al.The common YAP activation mediates corneal epithelial regeneration and repair with different-sized woundsnpj Regenerative Medicine2021ISSN 2057-3995
Gurusamy, Malarvizhi et al.G-protein-coupled receptor P2Y10 facilitates chemokine-induced CD4 T cell migration through autocrine/paracrine mediatorsNature Communications2021ISSN 2041-1723
Tocci, Piera et al.β-arrestin1/YAP/mutant p53 complexes orchestrate the endothelin A receptor signaling in high-grade serous ovarian cancerNature Communications2019ISSN 2041-1723
Eliazer, Susan et al.Wnt4 from the Niche Controls the Mechano-Properties and Quiescent State of Muscle Stem CellsCell Stem Cell2019ISSN 1875-9777
Hu, Jianjun et al.Opposing FlnA and FlnB interactions regulate RhoA activation in guiding dynamic actin stress fiber formation and cell spreadingHuman Molecular Genetics2017ISSN 1460-2083
Wang, Wenqi et al.AMPK modulates Hippo pathway activity to regulate energy homeostasisNature Cell Biology2015ISSN 1476-4679
Gray, Jason D. et al.LRP6 exerts non-canonical effects on Wnt signaling during neural tube closureHuman Molecular Genetics2013ISSN 0964-6906


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 RhoC levels in lysates?

Answer 2:  Yes, the rhotekin-RBD protein (Cat. # RT01) can be used to examine activated (GTP-bound) RhoA, RhoB, or RhoC  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. 



If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com