protocols

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.EscherichiacoliTheE.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.ThemitochondriawereisolatedaccordingtoMeisingeretal.,2000.Inbrief,cellswerepelletedbycentrifugation(3.000g,5min),washedwithdistilledwaterandresuspendedunderslowlyshakingfor20minat30°Cin2ml/gDTTbuffer(100mMTris-H2SO4pH9.4,10mMDTT).Afterwashingwithzymolyasebuffer(1.2Msorbitol;20mMpotassiumphosphate,pH7.4)cellswereincubatedwith5mg/g(w/v)Zymolyase-20T(SeikagakuKogyoCo.)in7ml/gzymolyasebufferfor45minat30°Cforconversationintospheroblasts.Homogenizationwascarriedoutby15strokesinaglass-Teflonpotterin6.5ml/gice-coldhomogenisationbuffer(0.6Msorbitol;10mMTris-HCl,pH7.4;1mMEDTA,1mMPMSF;0.2%(w/v)bovineserumalbumin).Thishomogenatewasdilutedwith1volice-coldhomogenisationbufferandcelldebrisandnucleiremovedbycentrifugation(1500g,5min,4°C).Thesupernatantwasagaincentrifuged(3000g,5min,4°C)andthemitochondriainthesupernatantpelletedbyadditionalcentrifugation(12.000g,15min,4°C).ThepelletwaswashedwithSEM(250mMsucrose;1mMEDTA;10mMMOPS,pH7.2),againcentrifuged(12.000g,15min,4°C)andresuspendedinSEMtogiveafinalconcentrationof5mg/ml.Themitochondrialfractionwastreatedby10strokesinaglass-Teflonpotterandloadedontoathree-stepsucrosegradient(1.5ml60%,4ml32%,1.5ml23%,1.5ml15%(w/v)sucroseinEMbuffer(10mMMOPS,pH7.2;1mMEDTA).Aftercentrifugation(134.000g,1h,2°C)thepurifiedmitochondriawererecoveredfromthe60%/32%interface,dilutedwith2volSEMandcentrifuged(10.000g,2°C).Themitochondrialpelletwasresuspendedinureabuffer(8Murea,2Mthiourea,1%(w/v)CHAPS,20mMDTT,0.8%(v/v)carrierampholytes3-10,100mMTris-HCl,pH7.5,1mMEDTA,14µMPMSF)andtheproteinconcentrationdetermined.Proteinaliquots(100µg)werestoredat-20°C.Priortoseparationinthefirstdimension(IEF)thevolumeoftheureabufferwasadjustedto350µlandtheproteinstransferedbyin-gelrehydrationintotheIEFgels.1.3.Mouse1.3.1.KidneyKidneysofadultmice(14.5days)weredissected,washedin5volofice-cold20mMTris/HClpH7.8,10mMEDTA,2mMEGTA,2mMDTT,dried(SpeedVac)andresuspendedin