Trichloroacetic-Acid-(TCA)-Precipitation-of-Protei

ProtocolTrichloroaceticAcid(TCA)PrecipitationofProteinsAndrewJ.LinkandJoshuaLaBaerINTRODUCTIONCellortissueextractsofproteinsoftencontaininterferingsubstancesthatmustberemoved.Anumberofmethodsareavailableforconcentratingdiluteproteinsolutionsandsimultaneouslyremovingcontaminatingsubstances.Trichloroaceticacid(TCA)/acetoneprecipitation,asdescribedhere,isoftenusedforthispurpose.MATERIALSItisessentialthatyouconsulttheappropriateMaterialSafetyDataSheetsandyourinstitution’sEnvironmentalHealthandSafetyOfficeforproperhandlingofequipmentandhazardousmaterialsusedinthisprotocol.ReagentsAcetoneProteinsampleTrichloroaceticacid(TCA)(Sigma-AldrichT9159)Preparea100%(w/v)solutionbydissolving2.2gofTCAin1mLofH2O.Thenpreparea10%solutionbyadding0.1mLof100%TCAsolutionto0.9mLofH2O.Keepbothsolutionsicecold.EquipmentMicrocentrifugeVacuumevaporator(e.g.,SpeedVac)METHOD1.Add0.11volumesofice-cold100%TCAtotheproteinsample.2.Placeonicefor10min.3.Add500µLofice-cold10%TCAtothesample.4.Placeonicefor20min.5.Centrifugeat20,000gfor30min.6.Carefullyremovethesupernatant.Aspirationofthesupernatantwithapipetteoradisposabletransferpipetteworkswell.Avoiddisturbingtheproteinpellet.7.Add500µLofacetone.Verygentlyrockthetubeonceortwicetorinsethetubeandpellet.AdaptedfromProteomics:AColdSpringHarborLaboratoryCourseManual,byAndrewJ.LinkandJoshuaLaBaer.CSHLPress,ColdSpringHarbor,NY,USA,2009.Citeas:ColdSpringHarbProtoc;2011;doi:10.1101/pdb.prot5651©2011ColdSpringHarborLaboratoryPress993PROTOCOLSColdSpringHarborProtocols(typically5min).Thepelletisnowreadyforresuspensioninamassspectrometrycompatiblebuffer.