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protocols.docx

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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：Protocols(ProteomicsCoreFacilityBiocenterOuluDept.ofBiochemistryUniversityofOuluFinland)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.ChemicalsAbbreviationsAPSBisCHAPSDTTPMSFPonceauSSDSTCEPTEMEDTrisAmmoniumPersulfateNN`-Methylene-bisacrylamide3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate14-dithio-DL-threitolPhenylmethanesulfonylFluoride3-Hydroxy-4-(2-sulfo-4-[4-sulfophenylazo]phenylazo)-27-naphthalenedisulfonicacidsodiumsaltSodiumdodecylsulfateTris-(2-carboxyethyl)phosphinehydrochlorideNNN´N´-TetramethylethylenediamineTris-[hydroxymethyl]aminomethane1.Samplepreparation1.1.EscherichiacoliTheE.colistrainRB791wasgrowninLBmediumandcells(50-100ml)collectedatdifferentgrowthstages.Afteracentrifugation(10min8000rpm4°C)thecellswerewashedtwicewith2mlTE-PMSF(1mMEDTA0.1MTris14µMPMSFpH7.5).Thepelletwasthenresuspendedin3mlTE-PMSFdisruptedbyFrenchPresswith900PSI(62.1bar)andthecelllysatecentrifuged(30min15.300rpm4°C).Thesupernatantwascollectedandtheproteinconcentrationdeterminedwithacommerciallyavailablekit(RotiNanoquant)accordingtoBradford1976.Aliquotsof100µgweredried(SpeedVac)andstoredat-20°C.Priortoseparationinthefirstd