蛋白相互作用

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1、ThermoSSicientifitifiPicPierce蛋蛋实白相互作用的研究方法和实践罗莎RosaLuoPh.D.ApplicationScientistBiosciencesDivisionThermoFisherScientificChina酵母蛋白质相互作用图谱°Thickbluelinesrepresentliterature-derivedinteractionsfromPreBIND+MIPSintheHMS-PCIdataset.°Thinorangelinesrepresent

2、potentialnovelinteractions.CourtesyMDSProteomics2蛋白质相互作用技术GeneticChemical°TwoHybrid°Crosslinking°PhageDisplay°Label-transfer°MttiMutationallanalliysis°FBABEFeBABEmappingBiochemicalFluorescent°Immunoprecipitation(IP)°Immunofluorescencecolocalization°Co-

3、IiImmunoprecipititatition(Co-IP)°FRET(FluorescenceResonance°Pull-DownAssaysEnergyTransfer)°FarWestern3蛋白相互作用研究方法•免疫沉淀(IP)•富集、纯化、检测兴趣蛋白Prey•用抗体捕获抗原•免疫共沉淀(Co-IP)•富集、纯化、检测兴趣蛋白Bait•用抗体捕获抗原和互作蛋白Prey•Pull-Down•确定蛋白之间的互作BaitPrey•用标签蛋白捕获结合蛋白4传统的免疫沉淀(IP)+Steppy

4、1.ImmobilizeantibodySteppp2.AddsampleStep4.Eluteantibody:proteinStep3.Bindproteinandcomplexwashawayunboundprotein+琼脂介质ProteinA/G抗原5传统的免疫沉淀碰到的困难IP)问题•选择适合的树脂•取液困难EitherlossofbeadsorsolutionremainsEitherlossofbeadsorsolutionremainswithbeads,contaminating

5、sample•抗体和抗原共洗脱AbAgAb+AgHeavyChainAntigenLightChain•抗体被破坏6免疫沉淀(Immunoprecipitation)AntigenPtiProteinA/G7IgG结合蛋白比较RecombinantRecombinantRecombinantRecombinantProteinAProteinGProteinA/GProteinL天然来源StaphylococcusStreptococcusN/APeptostreptococcaureususmag

6、nus产品来源E.coliE.coliE.coliE.coli分子量44,60021,60050,46035,800SDS-PAGE45kDa45kDa32kDa32kDa40-45kDa36kDa36kDa表观分子量IgG4264结合位点数量白蛋白结合位点NoNoNoNo最佳反应pH828.255-828.2757.5Ig结合位点FcFcFcVL-kappa8Pain:SelectingIgGbindingresin•ProteinA和ProteinG对不同来源和不同亚型的抗体结合能力不同.Ant

7、igen•ProteinA•只与Fc区域结合•对于IgG1和大鼠抗体结合力弱•ProteinG•结合Fc区和轻链PtiProteinProtein•对于IgA,IgM,IgE,等相对结合力较弱A/GG•ProteinA/GProteinA/G•蛋白质工程技术，将ProteinA的两个结合位点和ProteinG的4个抗体结合位点组ProteinG合到一起Protein•能够分别与ProteinA和G结合的抗体均A/GProtein可与之结合A9A/G+isthemostrobustIgGbinding

8、protienA/G+A+G+ControlLFTPDFTPDFTPDFTPDKey:L=LysateLoadAnti-PP2A(Mouse)FT=FlowThroughPD=SamplPllDlePullDownControl=Lysate+resin(noantibody)Anti-Cdk2(Rabbit)Anti-pp(53(Sheep)Conclusion:ThermoScientificPierceClassicIPKitiscompatiblewithmo