Rho GTPase activation assays.
The Rho GTPase family of signaling proteins controls a wide range of highly dynamic cellular processes. Activation of Rho GTPases can be investigated and quantified in cell extracts using so-called pull-down assays. Proteins that bind specifically to the activated form of the Rho GTPase are used to capture it onto a bead support. Western blotting of the captured samples with specific antibodies then allows for quantification of the level of Rho GTPase activation in the sample. This unit describes the techniques for preparing the reagents required for assays of RhoA, Rac, and Cdc42 and gives practical tips for the successful application of the assay in a range of situations.
Pellegrin
Stéphanie
S
Mammalian Cell Biology Laboratory, Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.
Mellor
Harry
H
D000818:Animals; D001681:Biological Assay; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D041701:Swiss 3T3 Cells; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein