1、Thermo Scientific Pierce,蛋白相互作用的研究方法,罗 莎,Rosa Luo Ph.D.,Application ScientistBiosciences Division,Thermo Fisher Scientific China,2,蛋白质相互作用技术,GeneticTwo HybridPhage DisplayMutational analysisBiochemicalImmunoprecipitation (IP)Co-Immunoprecipitation (Co-IP)Pull-Down AssaysFar WesternProprietary & Conf
2、idential,ChemicalCrosslinkingLabel-transferFeBABE mappingFluorescentImmunofluorescencecolocalizationFRET (Fluorescence ResonanceEnergy Transfer),3,General Purification of Proteins and,Interacting Proteins, Immunoprecipitation (IP) Co-IP, Pull-Down Assays,Proprietary & Confidential,4,蛋白相互作用研究方法 免疫沉淀
3、(IP) 富集、纯化、检测兴趣蛋白, 用抗体捕获抗原 免疫共沉淀 (Co-IP) 富集、纯化、检测兴趣蛋白,Prey, 用抗体捕获抗原和互作蛋白 Pull-Down 确定蛋白之间的互作 用标签蛋白捕获结合蛋白,Bait,Prey,Proprietary & Confidential,Bait,Prey,5,免疫沉淀 (Immunoprecipitation),Antigen,Protein,A/G,Proprietary & Confidential,6,Pain Points: 根据抗体选择树脂Protein A 和 Protein G 对不同来源,和不同亚型的抗体结合能力不同Protein
4、 A 只与 Fc 区域结合 对于IgG1 和大鼠抗体结合力弱,Antigen,Protein,GProtein G 结合Fc 区和部分轻链 对于IgA, IgM, IgE, 等相对结合力较弱,Proprietary & Confidential,ProteinG,ProteinA,7,Pain Points: 根据抗体选择树脂,Proprietary & Confidential,8,传统 Co-IP 技术 Known “bait” protein is used to pull out unknown “prey” target protein.+Step 2. Add sampleStep
5、 1. Immobilize antibody,Step 4. Elute bait:prey proteincomplexProprietary & Confidential,Step 3. Bind bait:prey protein complexand wash away unbound protein,9,Thermo Scientific 提供的四种IP/Co-IP 试剂盒,Proprietary & Confidential,Pierce Classic IP Kit,26146,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 抗体与抗原一起被洗脱,10
6、,Proprietary & Confidential,优点: Protein A/G 可与大部分抗体结合,Protein A/G 蛋白质工程技术，将Protein A的2个结合位点和Protein G的4,Antigen,11,个抗体结合位点组合到一起 能够分别与Protein A 和 G 结合的抗体均可与之结合Proprietary & Confidential,ProteinA/GProteinA/G,ProteinA/GProteinA/G,L,Protein A/G Plus的优势,A/G+,A+,G+,Control,Anti-PP2A (Mouse)Anti-Cdk2 (Rabb
7、it),FT PD,FT PD,FT PD FT PD,Key:L = Lysate LoadFT = Flow ThroughPD = Sample Pull DownControl = Lysate + resin(no antibody),Anti-p53 (Sheep)Conclusion利用Protein A/G 可以满足绝大部分抗体结合的需要.,12,Proprietary & Confidential,Pain Points: 实验的时间比较长 传统Batch法在同一个Ep管中操作 细胞裂解液和珠子和抗体在一个管子中进行孵育 通过离心沉淀珠子，分离固相和液相 取样不慎易导致样品损
8、失或污染 多孔材料的树脂会保留一定量的溶液,13,Proprietary & Confidential,用离心柱代替普通Ep管的优势 利用离心柱加速实验 利用带有滤膜的离心柱，可以通过离心分离固相和液相 洗涤的速度更快 避免样品的损失和相互干扰 带有底盖，可以满足样品孵育的需要 增加样品的得率 获得样品浓度高,14,Proprietary & Confidential,Pierce Classic IP Kit 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还原剂适用于大多数抗体使用方便
9、试剂盒提供全套试剂缺点 抗体不能再利用 抗体在Western Blot上被显色，影响结果,15,Proprietary & Confidential,抗体影响实验结果Immunoprecipitation of PP2A protein抗原被抗体覆盖,Ab,Ag,Ab + Ag,IgG Heavy Chain （55kD)PP2AIgG Light Chain （25kD）,二抗检测,抗体重链抗原抗体轻链,16,Proprietary & Confidential,Clean-Blot IP Detection Reagent Clean-Blot IP 检测试剂只结合未变性的一抗替代二抗用于
10、IP结果检测或检测含内源IgG的组织细胞裂解液去除变性的抗体对Western Blotting结果的干扰,Ab,Ag,Ab + Ag,Ab,Ag,Ab + Ag,二抗检测,抗体重链抗原抗体轻链,Clean-Blot IP 检测试剂,抗体重链抗原抗体轻链,17,Proprietary & Confidential,Clean-Blot IP Detection Reagent让IP结果更清晰,Clean-Blot IP Detection Reagent (HRP),GAM-HRP, p53 IP: Anti-p53 antibody (2 g) A431 total cell extract
11、(500 g) Protein A Agarose Resin, Western blot: Mouse anti-p53 antibody (BD Biosciences) (1 g/ml) Clean-Blot IP Detection Reagent (HRP)or Goat Anti-Mouse HRP (Product # 31430, 0.16 g/ml) SuperSignal West Pico ChemiluminescentSubstrate (Product # 34080).,18,Proprietary & Confidential,Clean-Blot IP 检测试
12、剂 Clean-Blot IP Detection Reagent (HRP) Product # 21230 Package Size: 2.5 mL Kit (HRP) Product # 21232 Clean-Blot IP Detection Reagent (HRP) 2.5 ml StartingBlock T20 (TBS) Blocking Buffer 1L Pierce ECL Detection Reagent 1, Peroxide Solution 125 ml Pierce ECL Detection Reagent 2, Luminol Enhancer Sol
13、ution 125 ml Clean-Blot IP Detection Reagent (Alkaline Phosphatase) Product # 21233 Package Size: 2.5 mLReferences：Journal of Biological Chemistry, 2010, Vol. 285, 5896-5906Molecular Biology of the Cell, 2009, Vol. 20, 43134323Science Signaling, 2008, Vol. 1, 43134323, ra17,19,Proprietary & Confiden
14、tial,Seize X抗体耦联,Pierce Crosslink,IP Kit,Bead,Protein Aor G,DSS交联剂,Bead,Protein Aor G,未结合抗体试剂盒组成DSS 交联剂：将抗体共价耦联到琼脂固相化的Protein A或G上,抗体被共价结合到蛋白A，G上Protein A/G Plus Agarose, 0.55mLDSS Crosslinker, 8 x 2mgCoupling Buffer (20X), 25mLIP Lysis/Wash Buffer, 2 x 50mLConditioning Buffer (100X), 5mLTris-Buffer
15、ed Saline (20X), 25mLElution Buffer, 50mLLane Marker Sample Buffer (5X), 5mLControl Agarose Resin, 2mLSpin Columns and Collection Tubes,20,Proprietary & Confidential,Pierce Crosslink IP Kit 26147特点 抗体非共价结合到蛋白 A/G Plus上 DSS 交联剂共价结合抗体和蛋白A/G Plus 抗体不被洗脱下来优点 高/中蛋白结合产量 可以使用纯化和未纯化的抗体 固定化 抗体抗原反复使用 抗体不被洗脱，不
16、影响抗原的检测 DSS交联可能需要优化实用，但操作步骤较多,21,Proprietary & Confidential,Seize Primary抗体耦联,Pierce Direct IP Kit,O,NH2,H,Bead,C,H,Bead,C,N,AminoLink Plus,Primary Antibody,Immobilized Antibody,SupportAminoLink Plus直接将抗体共价耦联到胺基活化的凝胶上,试剂盒组成,SupportAminoLink Plus Coupling Resin, 2mLCoupling Buffer (20X), 25mLQuenchin
17、g Buffer, 50mLWash Solution, 50mLSodium Cyanoborohydride Solution (5 M), 0.5mLIP Lysis/Wash Buffer, 2 x 50mLConditioning Buffer (100X), 5mLTris-Buffered Saline (20X), 25mLElution Buffer, 50mLLane Marker Sample Buffer (5X), 5mLControl Agarose Resin, 2mLSpin Columns and Collection Tubes,22,Proprietary
18、 & Confidential,Pierce Direct IP Kit 26148特点 无需蛋白A/G，抗体直接共价结合在基质上 无需交联剂，无需优化交联步骤 抗体不被洗脱下来优缺点 高/中蛋白结合产量 固定化 抗体抗原反复使用 抗体不被洗脱，不影响抗原的检测 抗体缓冲液不能够含有干扰成分（胺基基团）,TrisGlycineGelatinBSAOther proteins,23,Proprietary & Confidential,三种IP产品比较：Classic vs. Crosslink vs. Direct,24,ClassicProtein A,GProprietary & Conf
19、idential,CrosslinkAntibodies,交联剂,Direct,不同IP结果比较GFP Protein spiked into MOPC cell lysate,杂质少,无干扰,25,Proprietary & Confidential,Pierce Co-IP Kit,Bait,Prey,Highlights 与Direct IP kit原理相同 抗体直接与AminoLink bead交联 需要摸索适合蛋白质间相互作用和蛋白与抗体相互作用的条件,26,Proprietary & Confidential,Pierce HA- or c-Myc Tag IP/Co-IP Kit
20、,Anti-HAAnti-cMyc适用于过表达并含有HA/c-Myc蛋白标签的体系,偶联常用抗体,27,Proprietary & Confidential,Previous Seize kits versus new Pierce IP Kits,Previous KitsSeize Classic Protein ASeize Classic Protein GSeize Classic MammalianSeize X Protein ASeize X Protein GSeize X Mammalian (G)Seize PrimarySeize Primary MammalianPr
21、oFound Co-IPProFound Co-IP Mammalian,New KitsPierce Classic IP Kit(26146)Pierce Crosslink IP Kit(26147)Pierce Direct IP Kit(26148)Pierce Co-IP Kit(26149),28,Proprietary & Confidential,IP and co-IP方便可靠易于检测节约抗体,29,Proprietary & Confidential,Pulldown AssaysTag,Binding,molecule,ligand,Agarose,Bait,Prey,
22、bead,Highlights,Immobilization tags and ligands,30,Similar to a Co-IP a bait protein isused to pull down a prey proteinBait protein is immobilized throughtag rather than antibodyDifferent tags and immobilizationmethodsProprietary & Confidential,标签(on bait protein)6X HistidineGSTBiotin,配体(on bead)Nic
23、kel, cobalt谷胱甘肽Streptavidin,Pull-Down AssayGST tagged Bait Proteins 21516 用固定化的谷胱甘肽去结合融合了GST标签的Bait蛋白 用还原型的谷胱甘肽洗脱Bait:Prey复合物,PolyHis tagged Bait Proteins,21277, 用螯合了钴的固相支持物与融合了PolyHis标签的Bait蛋白进行结合 用咪唑洗脱Bait:Prey复合物,Biotin-labeled Bait Proteins,21115, 用固定化的链霉亲和素结合生物俗化的Bait蛋白 用低pH条件进行洗脱；仅将Prey蛋白洗脱下来，
24、而Bait蛋白依然结合在支持物上！,31,Proprietary & Confidential,Capturing Protein Interactions 使用交联剂 通过共价结合捕获相互作用（弱）的蛋白,32,Proprietary & Confidential,Crosslinkers (交联剂) 传统的交联剂 短的碳链，每一端有一个反应基团, Example: BS3,Na O,O,O,O,O SO,O,N,O,O,OO,N,OS OO Na, 将两个分子共价结合 可用于稳定蛋白-蛋白相互作用 每一端与一个特定的基团反应 (-NH2, -SH, -COOH, -C=O),33,Prop
25、rietary & Confidential,Diazirine Crosslinkers (双吖丙啶 ) 带有琥珀酰亚胺和双吖丙啶两个不同的功能基团 短化学链适于进行 fishing 实验 可对蛋白进行标记，并找出与之发生相互作用的蛋白In a protein mixture or inside cells 高度可控的交联操作 在适宜条件下，UV激活diazirine基团 短的化学链保证了交联是发生在真正发生相互作用的两个蛋白之间。经过活化的卡宾会和靠近的任何分子形成共价键，稳定结合 交联效率高于另一个用UV激活的交联剂aryl azide,34,Proprietary & Confiden
26、tial,Crosslinking标记,相互作用,空间靠近交联对蛋白相互作用的锁定是易于检测和可控的,35,Proprietary & Confidential,Label Transfer Reagents 标签转移 鉴定未知的相互作用的蛋白,36,Proprietary & Confidential,Label Transfer Reagents什么是标记转移试剂? 将交联剂和生物素化试剂相结合的产物 将生物素标记从纯化了的Bait蛋白上转移到未知的Prey蛋白什么研究会用到这类试剂? 已经有纯化好的Bait蛋白，而希望对与其发生相互作用的蛋白进行研究 在体外对酵母双杂交，Pull-Dow
27、nload Assay或者是Co-IP的结果进行验证,37,Proprietary & Confidential,Label Transfer标记相互作用空间靠近传递连接,38,Proprietary & Confidential,方法I: 验证相互作用,方法 II 质谱分析，寻找新蛋白,39,Proprietary & Confidential,SummaryGeneral purification of proteins and interacting proteins IP, Co-IP, Pulldown assaysCapturing protein interactions Crosslinkers UV activatable Diazirine crosslinkers Label Transfer Reagents,40,Proprietary & Confidential,更多信息 : & Confidential,更多信息Find Technical Handbooks & Confidential,谢谢！800 810 5118400 650 5118,43,Proprietary & Confidential,
