蛋白相互作用

Th Thermo Scientific S i tifi Pierce Pi蛋 蛋白相互作用的研究方法和实践 实罗 莎 Rosa Luo Ph.D. Application Scientist Biosciences Division Thermo Fisher Scientific China酵母蛋白质相互作用图谱ƒ Thick blue lines represent literature-derived interactions from PreBIND+MIPS in the HMS-PCI dataset. ƒ Thin orange lines represent potential novel interactions. Courtesy MDS Proteomics2蛋白质相互作用技术Genetic ƒ ƒ ƒ Two Hybrid Phage Display M t ti Mutational l analysis l i Chemical ƒ ƒ ƒ Crosslinking Label-transfer F BABE mapping FeBABE iBiochemical ƒ ƒ ƒ ƒ Immunoprecipitation (IP) C I Co-Immunoprecipitation i it ti (Co-IP) (C IP)Fluorescent ƒ ƒ Immunofluorescence colocalization FRET (Fluorescence Resonance Energy Transfer)Pull-Down Assays Far Western3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BaitPrey• Pull-Down• 确定蛋白之间的互作 • 用标签蛋白捕获结合蛋白BaitPrey4传统的免疫沉淀(IP)+Step p 1. Immobilize antibody y Step p 2. Add sample pStep 4. Elute antibody:protein complexStep 3. Bind protein and wash away unbound protein+ 琼脂介质5Protein A/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Either loss of beads or solution remains with beads, contaminating sample•抗体和抗原共洗脱 Ab Ag Ab+AgHeavy Chain AntigenLight Chain•抗体被破坏6免疫沉淀 (Immunoprecipitation)AntigenProtein P t i A/G7IgG结合蛋白比较Recombinant Protein A 天然来源 产品来源 分子量 SDS-PAGE SDS PAGE 表观分子量 IgG 结合位点数量 白蛋白结合位点 最佳反应pH Ig 结合位点Staphylococcus aureusRecombinant Protein GStreptococcusRecombinant Protein A/GN/ARecombinant Protein LPeptostreptococc us magnusE. coli 44,600 45 kDa 4 No 82 8.2 FcE. coli 21,600 32 kDa 2 No 5 FcE. coli 50,460 40 45 kDa 40-45 6 No 582 5-8.2 FcE. coli 35,800 36 kDa 4 No 75 7.5 VL-kappa8Pain: Selecting IgG binding resin• 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 A• • 只与 Fc 区域结合 对于IgG1 和大鼠抗体结合力弱Antigen•Protein G• • 结合Fc F 区和轻链 对于IgA, IgM, IgE, 等相对结合力较弱Protein P t i A/GProtein G•Protein A/G• 蛋白质工程技术，将Protein A 的两个结 合位点和Protein G的4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Protein G Protein A/G Protein A9A/G+ is the most robust IgG binding protienA/G+L FT PDA+FT PDG+ControlKey: L = Lysate Load FT = Flow Through PD = S Sample l P Pull ll Down D Control = Lysate + resin (no antibody)FT PD FT PDAnti-PP2A (Mouse) Anti-Cdk2 (Rabbit) ( ) Anti-p53 p ( (Sheep) p)Conclusion: Thermo Scientific Pierce Classic IP Kit is compatible with most IgG species isotypes.10Antibody Binding Protein SpecificityPierce 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取液困难——离心柱的解决方案p•Thermo Scientific Pierce Spin Columns•Thermo Scientific Pierce IP Kits contain convenient spincolumns•Flip top cap for easy handling•Screw cap for longer storage•No decanting or transferring ofwashes or samplesg g p g•Centrifuging provides faster washing•Tapered bottom provides improvedsample recovery (smaller volumesand more concentrated sample recovery)p y)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–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，影响结果抗体影响实验结果I i i i f PP2A iImmunoprecipitation of PP2A protein AbAgAb + Ag抗原被抗体覆盖IgG Heavy Chain （55kD)抗体重链抗原PP2AIgG Light Chain （25kD ）抗体轻链二抗检测Clean-Blot™ IP Detection Reagent•Clean-Blot™ IP 检测试剂•只结合未变性的一抗只结未变性抗•替代二抗•用于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 结果的干扰抗体重链Ab Ag Ab + AgAb Ag Ab + Ag抗体重链抗原抗体轻链抗原抗体轻链二抗检测Clean-Blot ™IP 检测试剂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 µg)A431total cell extract (500µg) Western blot :•Mouse anti-p53 antibody (BD Biosciences) (1 µg/ml)•Clean-Blot IP Detection Reagent (HRP)•A431 total cell extract (500 µg) •Protein A Agarose Resinor•Goat Anti-Mouse HRP (Product # 31430, 0.16 µg/ml)•SuperSignal West Pico ChemiluminescentSubstrate (Product # 34080).Clean-Blot™ IP 检测试剂•Clean-Blot™ IP Detection Reagent (HRP)•Product # 21230•Package Size: 2.5 mLPackage Size:25mL•Kit (HRP) Product # 21232Clean Blot IP Detection Reagent (HRP) 2.5 ml•Clean-Blot™IP Detection Reagent(HRP)2.5ml•StartingBlock T20 (TBS) Blocking Buffer 1L•Pierce ECL Detection Reagent 1, Peroxide Solution 125 ml•Pierce ECL Detection Reagent 2, Luminol Enhancer Solution 125 mlg,•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, 4313–4323Science Signaling, 2008, Vol. 1, 4313–4323, ra17Science Signaling2008Vol143134323ra17Seize® X 抗体耦联26147Pierce Crosslink IP KitBeadProtein A or GDSS 交联剂BeadProtein Aor G未结合抗体抗体被共价结合到蛋白A ，G 上试剂盒组成Protein A/G Plus Agarose, 0.55mL DSS Crosslinker, 8 x 2mg Coupling Buffer (20X), 25mL IP L i /W h B ff 250L DSS 交联剂：将抗体共价耦联到琼脂固相化的IP Lysis/Wash Buffer, 2 x 50mL Conditioning Buffer (100X), 5mL Tris-Buffered Saline (20X), 25mL Elution Buffer, 50mL Protein A 或G 上,Lane Marker Sample Buffer (5X), 5mL Control Agarose Resin, 2mLSpin Columns and Collection Tubes26147 Pierce Crosslink IP Kit特点抗体非共价结合到蛋白A/G Plus上•A/G Plus•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•固定化抗体反复使用•抗体不被洗脱，不影响抗原的检测•DSS交联可能需要优化实用，但操作步骤较多实用但操作步骤较多Seize® Primary 抗体耦联26148Pierce Direct IP KitONH 2H CHBeadCBeadNAminoLink PlusSupportPrimary AntibodyImmobilized AntibodySupportAminoLink AminoLink Plus Coupling Resin , 2mL Coupling Buffer (20X), 25mL Quenching Buffer, 50mL Wash Solution,50mL 试剂盒组成Plus直接将抗体共价耦联到胺基活化的凝胶上Wash Solution, 50mLSodium Cyanoborohydride Solution (5 M), 0.5mL IP Lysis/Wash Buffer, 2 x 50mL Conditioning Buffer (100X), 5mL Tris-Buffered Saline (20X), 25mL (),Elution Buffer, 50mLLane Marker Sample Buffer (5X), 5mL Control Agarose Resin, 2mLSpin Columns and Collection Tubes pPierce 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）¾Tris¾Glycine¾Gelatin¾BSA¾Other proteins三种IP产品比较：Classic vs. Crosslink vs. Direct Classic Crosslink DirectProtein A,G Antibodies交联剂不同IP结果比较GFP Protein spiked into MOPC cell lysate杂质少无干扰三种IP方法比较Traditional Pierce Pierce Pierce FeatureIP Method Classic IP Kit Crosslink IP Kit Direct IP KitUses high binding capacity resin Variable Yes+)Yes+)Yes(Protein A/G (Protein A/G (AminoLink+)Crosslinker mediatedimmobilizationNo No Yes NoRequires purified antibody inamine‐free and protein‐freestorage solutionNo No No YesAntibody covalently attached toresinNo No Yes Yes Antibody is oriented Yes Yes Yes No Antibody elutes with antigen Yes Yes No NoAntigen recovery method Boiling w/ SDS(Low pH)Low pH elution(Boiling w/Low pH elution Low pHelutionSDS)Relative antigen recovery Variable Highest High High Immobilized antibody can be No No Possible Possible reusedPierce Co-IP Kit 26149BaitPreyg gHighlights•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Pierce HA-or c-Myc Tag IP/Co-IP Kit 23610/20Anti-HA Anti-cMyc 偶联常用抗体y适用于过表达并含有HA/c-Myc 蛋白标签的体系Previous Seize kits versus new Pierce IP KitsPrevious KitsNew KitsS i Cl i P t i APierce Classic IP Kit(26146)}Seize Classic Protein A Seize Classic Protein G Seize Classic Mammalian Pierce Crosslink IP Kit}Seize X Protein A Seize X Protein G(26147)Pi Di t IP KitSeize X Mammalian (G)Seize Primary}Pierce Direct IP Kit (26148)Seize Primary Seize Primary Mammalian ProFound Co IP}Pierce Co-IP Kit(26149)Co-IP ProFound Co-IP MammalianIP and co-IP方便可靠易于检测节约抗体Capturing Protein Interactions•使用交联剂•通过共价结合捕获相互作用（弱）的蛋白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。