Nonradioactive methods for detecting activation of Ras-related small G proteins.
Ras-related small GTPases serve as critical regulators for a wide range of cellular signaling pathways and are activated by the conversion of the GDP-bound state to the GTP-bound conformation. Until recently, measurement of the GTP-bound active form of Ras-related G proteins involved immunoprecipitation of 32P-labeled protein followed by separation of the labeled GTP/GDP bound to GTPase. A new method based on the large affinity difference of the GTP- and GDP-bound form of Ras proteins for specific binding domains of effector proteins in vitro has been developed. By using glutathione S-transferase (GST) fusion proteins containing these binding domains, the GTP-bound form of the GTPase can be precipitated from cell lysates. In principle, this method can be used for all members of the Ras superfamily. Here we describe a general procedure to monitor the GTP-bound form of Ras-related GTPases.
Andres
Douglas A
DA
Department of Molecular and Cellular Biochemsitry, University of Kentucky College of Medicine, Lexington, USA.
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005982:Glutathione Transferase / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D020559:Monomeric GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D011233:Precipitin Tests / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction; D018631:ras Proteins / Q000032:analysis / Q000378:metabolism
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins