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Chapter3Methods陈建国新生物楼518chenjg@pku.edu.cnThelong-rangegoal•Completeunderstandingofwhattakeplaseinsideacellasitrespondstoitsenvironmentandinteractswithitsneighbors.•Howthecellsuccessfullymanagesthisstaggeringnumberofvariablesandhowitcanchoosesamonganalmostunlimitednumberofpossibilitiesinperformingitsdiversebiologicaltasks.•Howgenesandproteinsoperatetolaythefoundationsforlife.IsolatingcellandgtowingthemincultureCellsCanBeIsolatedfromaTissueSuspensionandSeparatedintoDifferentTypesCompositionofaTypicalMediumSuitablefortheCultivationofMammalianCellsAMINOACIDSVITAMINSSALTSMISCELLANEOUSPROTEINSArgininebiotinNaClglucoseinsulinCystinecholineKClpenicillintransferrinGlutaminefolateNaH2PO4streptomycingrowthfactorsHistidinenicotinamideNaHCO3phenolredIsoleucinepantothenateCaCl2wholeserumLeucinepyridoxalMgCl2LysinethiamineMethionineriboflavinPhenylalanineThreonineTrytophanTyrosineValineEucaryoticCellLinesAreaWidelyUsedSourceofHomogeneousCellsSomeCommonlyUsedCellLinesCELLLINE*CELLTYPEANDORIGIN3T3fibroblast(mouse)BHK21fibroblast(Syrianhamster)MDCKepithelialcell(dog)HeLaepithelialcell(human)PtK1epithelialcell(ratkangaroo)L6myoblast(rat)PC12chromaffincell(rat)SP2plasmacell(mouse)COSkidney(monkey)293kidney(human);transformedwithadenovirusCHOovary(chinesehamster)DT40lymphomacellforefficienttargetedrecombination(chick)R1embryonicstemcells(mouse)E14.1embryonicstemcells(mouse)H1,H9embryonicstemcells(human)Figure8-5MolecularBiologyoftheCell(©GarlandScience2008)Figure8-6MolecularBiologyoftheCell(©GarlandScience2008)CellsCanBeFusedTogethertoFormHybridCellsTheproductionofhybridcells.Humancellsandmousecellsarefusedtoproduceheterocaryons,whicheventuallyformhybridcells.Theseparticularhybridcellsareusefulformappinghumangenesonspecifichumanchromosomes.HybridomaCellLinesProvideaPermanentSourceofMonoclonalAntibodiesFractionationofCellsOrganellesandMacromoleculesCanBeSeparatedbyUltracentrifugationA.ThetechniqueofdifferentialcentrifugationCellfractionationbycentrifugation.Repeatedcentrifugationatprogressivelyhigherspeedswillfractionatehomogenatesofcellsintotheircomponents.lowspeed1000timesgravityfor10minutesmediumspeed20,000timesgravityfor20minuteshighspeed80,000timesgravityfor1hourveryhighspeed150,000timesgravityfor3hourComparisonofvelocitysedimentationandequilibriumsedimentation.Figure8-73MolecularBiologyoftheCell(©GarlandScience2008)Figure8-18aMolecularBiologyoftheCell(©GarlandScience2008)AnalyzingProteinsSDS-PAGEFigure8-18bMolecularBiologyoftheCell(©GarlandScience2008)Figure8-19MolecularBiologyoftheCell(©GarlandScience2008)Figure8-22MolecularBiologyoftheCell(©GarlandScience2008)TwodimensionalgelelectrophoresisFigure8-23MolecularBiologyoftheCell(©GarlandScience2008)Figure8-21MolecularBiologyoftheCell(©GarlandScience2008)ProteinInteraction•Yeasttwohybrid•Co-IP•GSTPull-down•Fluorescenceresonanceenergytransfer（FRET)Figure8-24MolecularBiologyoftheCell(©GarlandScience2008)Yeasttwo-hybridFigure8-16MolecularBiologyoftheCell(©GarlandScience2008)GSTpull-downFigure8-26MolecularBiologyoftheCell(©GarlandScience2008)FRETFigure8-64MolecularBiologyoftheCell(©GarlandScience2008)StudyinggeneexpressionandfunctionFigure8-65MolecularBiologyoftheCell(©GarlandScience2008)Hypertrophyandinvadingtumor-likerosettephenotypeofkap3cKOmouseRNAiFigure8-67aMolecularBiologyoftheCell(©GarlandScience2008)Figure8-67bMolecularBiologyoftheCell(©GarlandScience2008)THANKS!