Determination of GTP loading on Rho
Determination of GTP loading on Rho
Publisher Summary This chapter discusses the determination of guanosine 5'-triphosphate (GTP) loading on Rho. Rho is a member of the Ras superfamily of low molecular weight GTPase that is implicated in the regulation of actin cytoskeleton organization. The chapter presents an affinity precipitation assay for endogenous GTP-loaded (active) Rho and also describes the expression and purification of Rhotekin Rho-binding domain (GST-TRBD). The Rho-binding domain (RBD) from Rhotekin (one of the Rho effectors) has been reported to inhibit both the intrinsic and the GTPase-activating protein (GAP)-enhanced GTPase activity of Rho. Because Rho effectors interact only with GTP- Rho, the Rhotekin Rho-binding domain (TRBD) has been used for affinity precipitate endogenous GTP-Rho from cell lysates. The chapter also describes the detection of Rho by western immunoblotting and quality control of TRBD and positive control for Rho Assay. The chapter concludes with a discussion of the quality control of TRBD and positive control for Rho assay.
- State University of New York at Potsdam United States
- State University of New York United States
rho GTP-Binding Proteins, Binding Sites, Recombinant Fusion Proteins, Blotting, Western, Intracellular Signaling Peptides and Proteins, Guanosine Triphosphate, Carrier Proteins, Peptide Fragments, Protein Binding
rho GTP-Binding Proteins, Binding Sites, Recombinant Fusion Proteins, Blotting, Western, Intracellular Signaling Peptides and Proteins, Guanosine Triphosphate, Carrier Proteins, Peptide Fragments, Protein Binding
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