JournalofVisualizedExperimentswww.jove.comCopyright©2012CreativeCommonsAttribution-NonCommercialLicensePage1of2MaterialsListfor:AffinityPrecipitationofActiveRho-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:10.3791/3932MaterialsNameCompanyCatalogNumberCommentspGEX-RhoA(G17A)AddgeneWillbeavailablesoonGeneratedinthelabofDr.KeithBurridge(UniversityofNorthCarolina)(ref3)DH5αInvitrogen18258-012Store-80°CSOCmediumBioShopCanadaSOC001.10LBBioShopCanadaLBL407.1KeepsterileGlycerolBioShopCanadaGLY002.1AmpicillinBioShopCanadaAMP201.25Storestockat-20°CAgarBioShopCanadaAGR001.500IPTG(IsopropylB-D-thiogalacto-pyranoside)Calbiochem420322Storestocksolutionat-20°CGlutathioneSepharosebeadsGEHealthcare17-0756-01PBS10xSigma-AldrichD1408HepesSigma-AldrichH4034NaClBioShopCanadaSOD001.10MgCl2Sigma-AldrichM-9272AddjustbeforeuseTX-100Sigma-AldrichX100DTTOmniPur,EMDMillipore3860AddjustbeforeusePMSFSigma-AldrichP-7626AddjustbeforeuseProteaseInhibitor50xBDBiosciences51-21426ZAddjustbeforeuseCompleteMini,EDTA-free10xRocheGroup11836170001AddjustbeforeuseLaemmliSampleBuffer2xBio-Rad161-0737β-mercapt–thanolSigma-AldrichM7154AddjustbeforeuseCoomassieBrilliantBlueBio-RadR-250AceticAcidCALEDON1000-1MethanolCALEDON6701-7Bio-RadProteinAssayBio-Rad500-0114BLUeyeladderFroggaBioPM008-0500Table1.ReagentsusedRC-5BcentrifugeSorvall,ThermoScientificSS-34RotorUseat4°CCentrifugeSorvall,ThermoScientificST-16RUseat4°CMicrocentrifugeEppendorf5417RUseat4°CBacterialshakerINFORSHTEcotronAGCH-1403BottmingenUseat37°Corat22°CJournalofVisualizedExperimentswww.jove.comCopyright©2012CreativeCommonsAttribution-NonCommercialLicensePage2of2SonicatorBransonSonifier-450UseatRTRotatorGlas-Col099ACR4012Useat4°CTable2.Specificequipmentsused
