JournalofVisualizedExperimentswww.jove.comCopyright©2012CreativeCommonsAttribution-NonCommercialLicenseMarch2012|61|e3932|Page1of5VideoArticleAffinityPrecipitationofActiveRho-GEFsUsingaGST-taggedMutantRhoProtein(GST-RhoA(G17A))fromEpithelialCellLysatesFaizaWaheed1,2,PamelaSpeight1,2,QinghongDan1,2,RafaelGarcia-Mata3,KatalinSzaszi1,21KeenanResearchCentre,LiKaShingKnowledgeInstitute,St.Michael'sHospital2DepartmentofSurgery,UniversityofToronto3DepartmentofCellandDevelopmentalBiology,UniversityofNorthCarolinaatChapelHillCorrespondenceto:KatalinSzasziatszaszik@smh.caURL:http://www.jove.com/video/3932DOI:doi:10.3791/3932Keywords:MolecularBiology,Issue61,RhoFamilySmallGTPases,Guanine-nucleotideexchangefactor(GEFs),AffinityPrecipitationAssay,expressionofproteinsinE.Coli,PurificationofGST-taggedProtein,microbeadassayDatePublished:3/31/2012Citation:Waheed,F.,Speight,P.,Dan,Q.,Garcia-Mata,R.,Szaszi,K.AffinityPrecipitationofActiveRho-GEFsUsingaGST-taggedMutantRhoProtein(GST-RhoA(G17A))fromEpithelialCellLysates.J.Vis.Exp.(61),e3932,doi:10.3791/3932(2012).AbstractProteinsoftheRhofamilyofsmallGTPasesarecentralregulatorsofthecytoskeleton,andcontrolalargevarietyofcellularprocesses,includingcellmigration,geneexpression,cellcycleprogressionandcelladhesion1.RhoproteinsaremolecularswitchesthatareactiveinGTP-boundandinactiveinGDP-boundstate.TheiractivationismediatedbyafamilyofGuanine-nucleotideExchangeFactor(GEF)proteins.Rho-GEFsconstitutealargefamily,withoverlappingspecificities2.AlthoughalotofprogresshasbeenmadeinidentifyingtheGEFsactivatedbyspecificsignals,therearestillmanyquestionsremainingregardingthepathway-specificregulationoftheseproteins.ThenumberofRho-GEFsexceeds70,andeachcellexpressesmorethanoneGEFprotein.Inaddition,manyoftheseproteinsactivatenotonlyRho,butothermembersofthefamily,contributingfurthertothecomplexityoftheregulatorynetworks.Importantly,exploringhowGEFsareregulatedrequiresamethodtofollowtheactivepoolofindividualGEFsincellsactivatedbydifferentstimuli.Hereweprovi...
