ThSitifiPiThermoScientificPierce蛋白相互作用的研究方法和实践蛋实罗莎罗莎RosaLuoPh.D.ApplicationScientistBiosciencesDivisionThermoFisherScientificChina酵母蛋白质相互作用图谱�Thickbluelinesrepresentliterature-derivedinteractionsfromPreBIND+MIPSintheHMS-PCIdataset.�Thinorangelinesrepresentpotentialnovelinteractions.CourtesyMDSProteomics2蛋白质相互作用技术�TwoHybrid�CrosslinkingGeneticChemical�PhageDisplayMttilli�Label-transferFBABEi�Mutationalanalysis�FeBABEmappingBiochemicalFluorescent�Immunoprecipitation(IP)CIiitti(CIP)�ImmunofluorescencecolocalizationBiochemicalFluorescent�Co-Immunoprecipitation(Co-IP)�Pull-DownAssays�FRET(FluorescenceResonanceEnergyTransfer)�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•富集、纯化、检测兴趣蛋白•用抗体捕获抗原Prey•免疫共沉淀(Co-IP)•富集、纯化、检测兴趣蛋白•用抗体捕获抗原和互作蛋白PreyBait•用抗体捕获抗原和互作蛋白•Pull-DownPrey•确定蛋白之间的互作•用标签蛋白捕获结合蛋白PreyBait4传统的免疫沉淀(IP)+Step2.AddsampleStep1.ImmobilizeantibodypppyStep4.Eluteantibody:proteincomplexStep3.Bindproteinandwashawayunboundprotein+琼脂介质抗原5琼脂介质ProteinA/G抗原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,contaminatingsample•抗体和抗原共洗脱Ab+AgAbAgHeavyChainAntigenAb+AgAbAgLightChain•抗体被破坏6免疫沉淀(Immunoprecipitation)AntigenPtiProteinA/G7IgG结合蛋白比较RecombinantProteinARecombinantProteinGRecombinantProteinA/GRecombinantProteinL天然来源StaphylococcusStreptococcusN/APeptostreptococc天然来源StaphylococcusaureusStreptococcusN/APeptostreptococcusmagnus产品来源E.coliE.coliE.coliE.coli分子量44,60021,60050,46035,800SDSPAGE45kDa32kDa4045kDa36kDaSDS-PAGE表观分子量45kDa32kDa40-45kDa36kDaIgG结合位点数量4264结合位点数量白蛋白结合位点NoNoNoNo最佳反应pH82558275最佳反应pH8.255-8.27.5Ig结合位点FcFcFcVL-kappa8Pain:SelectingIgGbindingresinAntigen•ProteinA和ProteinG对不同来源和不同亚型的抗体结合能力不同.•ProteinA•只与Fc区域结合PtiProtein•对于IgG1和大鼠抗体结合力弱•ProteinG结合F区和轻链ProteinA/GProteinG•结合Fc区和轻链•对于IgA,IgM,IgE,等相对结合力较弱•ProteinA/GProteinGProteinA/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ProteinA/GGProteinA合到一起•能够分别与ProteinA和G结合的抗体均可与之结合9可与之结合A/G+isthemostrobustIgGbindingprotienA/G+A+G+ControlLFTPDFTPDFTPDFTPDKey:L=LysateLoadFT=FlowThroughPDSlPllDAnti-PP2A(Mouse)LFTPDFTPDFTPDFTPDPD=SamplePullDownControl=Lysate+resin(noantibody)Anti-Cdk2(Rabbit)()Anti-p53(Sheep)p(p)Conclusion:ThermoScientificPierceClassicIPKitiscompatiblewithmostIgGspeciesisotypes.10AntibodyBindingProteinSpecificity11PierceClassicIPKit26146样品样品（如细胞裂解液）•抗体通过蛋白A/G与基质结合•用于从样品中纯化抗原用于从样品中纯化抗原•抗体与抗原一起被洗脱12取液困难——离心柱的解决方案•ThermoScientificPierceSpinColumnsp•ThermoScientificPierceIPKitscontainconvenientspincolumns•Fliptopcapforeasyhandling•Screwcapforlongerstorage•Nodecantingortransferringofwashesorsamples•Centrifugingprovidesfasterwashingggpg•Taperedbottomprovidesimprovedsamplerecovery(smallervolumesandmoreconcentratedsamplerecovery)13py)PierceClassicIPKit特点结果稳定–Pierce使用离心管形式，使用方便，避免琼脂/抗体及样品的损失，降低非特异性快速–一小时之内完成洗脱条件温和–回收抗原不使用强烈的去污剂和...
