Cat. # RT02
Product Uses Include
- Measurement of the GTP/GDP ratio of RhoA/B/C in vitro.
- Quantitation of GTP-RhoA/B/C from tissue and tissue culture cell lysates.
Material
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 these proteins 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 in a glutathione agarose bound format and is shipped lyophilized. The beads are colored for ease of use. This product is used in our RhoA pulldown activation assay Biochem Kit™ (Cat. # BK036). The GST-Rhotekin-RBD is also available as a free protein (Cat. # RT01).
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Figure 1. The brightly colored glutatione agarose beads in RT02 are easy to use. |
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-Rhotekin-RBD protein is ~85% pure (see Figure 2).
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Figure 2: GST-Rhotekin-RBD protein purity determination. A 20 µg sample of RT02 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue. The GST-Rhotekin-RBD protein runs at approximately 35 kDa. |
Biological Activity
The Rhotekin-RBD protein specifically recognizes and binds to the active, GTP-bound, form of Rho protein. It has a much lower affinity for the inactive, GDP-bound, form of Rho. Biological activity of Rhotekin-RBD protein is therefore determined by its selectivity for GTP-Rho protein. The assay for monitoring the GTP-bound form of Rho is a protein pulldown assay using RT02 beads and a human platelet extract loaded with GTPγS (Cat. # BS01).
Examples of publications where this product was used:
Bi, Y. and Williams, J. A. (2005). A role for Rho and Rac in secretagogue induced amylase release by pancreatic acini. Am. J. Physiol. 289, C22-33
Ishii, S., Kihara, Y. and Shimizu, T. (2005). Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor. J. Biol. Chem. 280, 9083-9087.
Saito, S., Liu, X. F., Kamijo, K., Raziuddin, R., Tatsumoto, T., Okamoto, I., Chen, X., Lee, C. C., Lorenzi, M. V., Ohara, N. et al. (2004). Deregulation and mislocalization of the cytokinesis regulator ECT2 activate the Rho signaling pathways leading to malignant transformation. J. Biol. Chem. 279, 7169-7179.
Wang, Q., Liu, M., Kozasa, T., Rothstein, J. D., Sternweis, P. C. and Neubig, R. R. (2004). Thrombin and lysophosphatidic acid receptors utilize distinct rhoGEFs in prostate cancer cells. J. Biol. Chem. 279, 28831-28834.
Wang, S. E., Wu, F. Y., Shin, I., Qu, S. and Arteaga, C. L. (2005). Transforming growth factor β (TGF-β)-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-β function. Mol. Cell. Biol. 25, 4703-4715.
Zhang, X. F., Schaefer, A. W., Burnette, D. T., Schoonderwoert, V. T. and Forscher, P. (2003). Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow. Neuron 40, 931-944.
| Product description | Cat. # | Amount | Price & Order |
| Rhotekin-RBD protein agarose beads | RT02-A | 2 x 2 mg |
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| RT02-B | 6 x 2 mg |
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