2017_protein_characterization(1).pptVIP免费

1Proteincharacterization2017-10-12chunguangwang@tongji.edu.cn王春光2Gelelectrophoresis凝胶电泳3SDS-PAGE(SodiumDodecylSulfate十二烷基磺酸钠)Sodiumdodecylsulfate,CH3(CH2)10CH2-SO4-,Na+-mercaptoethanolHS-CH2CH2-OHDTT(dithiothreitol)Sthtoknowbytheway:4SDS-PAGE(PolyAcrylamideGelElectrophoresis聚丙烯酰胺凝胶电泳)%GelMassRange750kDa-500kDa1020kDa-300kDa1210kDa-200kDa153kDa-100kDahttp://employees.csbsju.edu/hjakubowski/classes/ch331/Techniques/TechElectrophoresis.htmpolymerizeAcrylamideisneurotoxic!5VisualizationofproteinongelCoomassiebrilliantbluestainingSilverstaining银染考马斯亮蓝染色InvolvingthereductionofAg+toAg;10-50timessensitivethancoomassieblue.6IEF(isoelectricfocusinggel等电聚焦电泳)7Two-dimensionalgel(2D-gel)8Westernblot（免疫印迹）SDS-PAGEWesternblot9Massspectrometry质谱m/zmolecularweight;error<100ppm.(http://www.piercenet.com)10http://upload.wikimedia.org/wikipedia/commons/1/1f/Mass_spectrometry_protocol.pngPowerfulmodernmassspectrometry11MethodstoquantifyproteinUVabsorbance:A=cl(Lambert-Beerlaw)absorbancecoefficientconcentrationlengthoflightpathGenerally,OD280=1for0.5~2mg/mlproteinStainingmethodsBiuretmethod,Lowrymethod,Bradfordmethod,etc.12Edmandegradationsequencing13PTHAminoAcidStandardResidueLysEdmandegradationsequencing14NCEdmansequencing=N-terminalsequencingNeedtocutproteinintolimitednumberoffragments.15ProteasedigestiontofragmentproteinstrypsinchymotrypsinpepsinGlu-C,Lys-C,Arg-C,Asp-N,etc;Cyanogenbromide(CNBr)C-terminiofTyr,Phe,andTrp.Leu,Met,Ala,Asp,andGluarecleavedatalowerrate.C-terminiofLysandArg.ChemicalcleavageattheC-terminusofMet.MainlyatC-terminiofPheandLeu.16NC17OverlappingtogetcompletesequenceTTCC18Three-dimensionalstructuredeterminationX-raycrystallographyNMR(NuclearMagneticResonance)EM(Electronicmicroscopy)Structureinsolution;Forsmallproteins(<20KD);Forlargeproteinsorproteinassemblies;Relativelylowresolution.19ToknowproteinpurityandsizeToknowproteinstructuresElectrophoresis(SDS-PAGE,IEF,2Dgel,etc.)MassspectrometryEdmandegradationsequencingX-raycrystallography,etc.ToquantifyproteinsUVabsorbance;roughstaining;etc.Commonlyusedtechniques20Youarereadytostudyproteins!