第1页共10页编号：时间：2021年x月x日书山有路勤为径，学海无涯苦作舟页码：第1页共10页Protocols(ProteomicsCoreFacility,BiocenterOulu,Dept.ofBiochemistry,UniversityofOulu,Finland)Abbreviations1.Samplepreparation1.1.Escherichiacoli1.2.Saccharomycescerevisiae1.2.1.Wholeintracellularextract1.2.2.Mitochondria1.3.Mouse1.3.1.Kidney1.3.2.Bones(calvariaandfemur)1.3.3.T-cellsfromspleen1.3.4.FibroblastcelllineNIH3T31.4.Human1.4.1.Serum1.4.2.Toothpulptissue2.IEF(Isoelectricfocusing)2.1.In-gelrehydration2.2.Samplecuploading3.SDS-PAGE(SDS-polyacrylamidegelelectrophoresis)3.1.CastingtheSDSgels3.1.1.2-DSDS-PAGE3.1.2.1-DSDS-PAGE3.2.Electrophoresis3.2.1.2-DSDS-PAGE3.2.2.1-DSDS-PAGE4.Proteindetection4.1.Silverstaining4.2.WesternBlotting5.Proteinidentification(massspectrometry)6.Troubleshooting7.Linkstootherprotocols8.References9.ChemicalsAbbreviationsAPSBisCHAPSDTTPMSFPonceauSSDSTCEPTEMEDTrisAmmoniumPersulfateN,N`-Methylene-bisacrylamide3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate1,4-dithio-DL-threitolPhenylmethanesulfonylFluoride3-Hydroxy-4-(2-sulfo-4-[4-sulfophenylazo]phenylazo)-2,7-naphthalenedisulfonicacidsodiumsaltSodiumdodecylsulfateTris-(2-carboxyethyl)phosphinehydrochlorideN,N,N´,N´-TetramethylethylenediamineTris-[hydroxymethyl]aminomethane1.Samplepreparation1.1.Escherichiacoli第2页共10页第1页共10页编号：时间：2021年x月x日书山有路勤为径，学海无涯苦作舟页码：第2页共10页TheE.colistrainRB791wasgrowninLBmediumandcells(50-100ml)collectedatdifferentgrowthstages.Afteracentrifugation(10min,8000rpm,4°C)thecellswerewashedtwicewith2mlTE-PMSF(1mMEDTA,0.1MTris,14µMPMSF,pH7.5).Thepelletwasthenresuspendedin3mlTE-PMSF,disruptedbyFrenchPresswith900PSI(62.1bar)andthecelllysatecentrifuged(30min,15.300rpm,4°C).Thesupernatantwascollectedandtheproteinconcentrationdeterminedwithacommerciallyavailablekit(RotiNanoquant)accordingtoBradford,1976.Aliquotsof100µgweredried(SpeedVac)andstoredat-20°C.Priortoseparationinthefirstdimension(IEF)theproteinswereresuspendedin350µlureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris/HCl,pH7.5,1mMEDTA,14µMPMSF)andtransferedbyin-gelrehydrationintotheIEFgels.1.2.Saccharomycescerevisiae1.2.1.WholeintracellularextractFortheanalysisofthefermentativegrowththeS.cerevisiaewildtypestrainBJ1991wascultivatedonrichYPD(1%(w/v)yeastextract,2%(w/v)peptone,and2%(w/v)D-glucose)andcollectedatanopticaldensity(600nm)of1.1.ForthestudyoftherespirationawildtypeculturewasgrowninYPDtoanopticaldensity(600nm)of1-2,thenpelleted,andtransferredtoYPGcontaining3%(v/v)glycerolinsteadofD-glucose.After16hcellswerecollected,centrifuged(10min,8000rpm,4°C)andwashedtwicewith2mlTE-PMSF(1mMEDTA,0.1MTris,14µMPMSF,pH7.5).Thenthepelletwasresuspendedin3mlTE-PMSFanddisruptedbyFrenchPresswith900PSI(62.1bar).Thecelllysatewascentrifugated(30min,15.300rpm,4°C)andthesupernatantcollected.Theproteinconcentrationwasdeterminedwithacommerciallyavailablekit(RotiNanoquant)accordingtoBradford,1976.Aliquotsof100µgweredried(SpeedVac)andstoredat-20°C.Priortoseparationinthefirstdimension(IEF)theproteinswereresuspendedin350µlureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris/HCl,pH7.5,1mMEDTA,14µMPMSF)andtransferedbyin-gelrehydrationintotheIEFgels.1.2.2.MitochondriaYeastcellsweregrownandcollectedasdescribedabove.Themitochondriawere...
