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1、ProteinElectrophoresisandSDS-PAGECai-xiaJinAssociateProfessorDepartmentofRegenerativeMedicineTongjiUniversityMedicalSchoolEmail:ObjectiveTounderstandtheprinciplesofproteinseparationbySDSpolyacrylamidegelelectrophoresis(SDS-PAGE).BeabletouseSDS-PAGEtechniquetoseparateproteins.Proteins are separated u
2、sing polyacrylamide gels rather than agarose gels,because the polyacrylamide matrix.A)have pore sizes similar to the sizes of proteins(DNA is usually larger in size)B)are much tighter than agarose gels,thus are able to resolve the smaller protein moleculesAQuickIntroductiontotheSDS-PAGEPrincipleWhat
3、isSDS-PAGE?SDS(Sodiumdodecylsulphate)isadetergentusedtodenatureproteinsandgivethemanegativecharge PAGE:PolyacrylamideGelElectrophoresis Itisatechniquetoseparateproteinsbytheirmolecularweight Priortoelectrophoresis,proteinsaretreatedwithadetergentsolution,thesodiumdodecylsulfate(SDS),andheatedUses tw
4、o phases of polyacrylamide:1)An upper stacking gel that is usually 4%acrylamide allows the proteins to migrate rapidly and concentrates them into uniform bands before hitting the denser gel 2)A lower separating gel that is of a higher percentage of acrylamide(usually 815%)separates according to mole
5、cular weightSDS-PAGETris-Cl SDS Tris-HClbuffer(pH:6.8and8.8)Acrylamide APS:Ammoniumpersulfate TEMEDWhatisinthegel?Whatisinthebuffer?SDS-PAGErunningbuffer MadewithTrisandglycine Thechlorineions(gel)migratemuchmorerapidlythentheglycineions(buffer)withtheproteinshavingamigrationspeedinbetweenthetwoions
6、proteinsbecometrappedinanarrowbandbetweenthetwoionfrontswhenelectrophoresisbegins.GelPreparation-1Procedure12%separatinggelReagents Volume(mL)H2O 4.340%Arc/Bis 31.5MTris-Cl(pH8.8)2.510%SDS 0.110%APS 0.1TEMED 0.0055%stackinggelReagents Volume(mL)H2O 2.3740%Arc/Bis 0.50.5MTris-Cl(pH6.8)110%SDS 0.0410%
7、APS 0.04TEMED 0.005GelPreparation-2Makeseparatinggelfirst,thenstackinggel Assoonastheelectriccurrentisapplied,theSDScoatedproteinsbegintheirjourneytowardsthepositiveelectrode.Smallerproteinsmigratemorequicklythanlargerproteinsthroughthepolyacrylamidepores1.Addrunningbuffer;2.30Vx1hour.Electropheresi
8、s-2VisualizingyourProteinafterElectrophoresis ThegelisstainedwithCoomassieBrilliantBlueR-250 Thisstainbindsspecificallytoproteinsandnoothermolecule,suchasDNA,carbohydrates,orlipids.Afterstaining,distinctbluebandsappearonthegelbasedonhowmuchproteinisinthatband Thelargertheamountofprotein,themoreintensethestainingResults