蛋白相互作用-ThermoFisher

Thermo Scientific Pierce Th S i tifi 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
Thick blue lines represent literature-derived interactions from PreBIND+MIPS in the HMS-PCI dataset. Thin orange lines represent potential novel interactions. Courtesy MDS Proteomics
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蛋白质相互作用技术
Genetic Two Hybrid Phage Display Mutational analysis M t ti l l i Biochemical Immunoprecipitation (IP) Co-Immunoprecipitation (C IP) C I i it ti (Co-IP) Pull-Down Assays Far Western FRET (Fluorescence Resonance Energy Transfer) Chemical Crosslinking Label-transfer FeBABE F BABE mapping i Fluorescent Immunofluorescence colocalization
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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
Bait
Prey
• Pull-Down
• 确定蛋白之间的互作 • 用标签蛋白捕获结合蛋白
Bait
Prey
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传统的免疫沉淀(IP)
+
Step 1. Immobilize antibody p y Step 2. Add sample p p
Step 4. Elute antibody:protein complex
Step 3. Bind protein and wash away unbound protein
+ 琼脂介质
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Protein 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+Ag
Heavy Chain Antigen
Light Chain
•
抗体被破坏
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免疫沉淀 (Immunoprecipitation)
Antigen
Protein P t i A/G
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IgG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 aureus
Recombinant Protein G
Streptococcus
Recombinant Protein A/G
N/A
Recombinant Protein L
Peptostreptococc us magnus
E. coli 44,600 45 kDa 4 No 8.2 82 Fc
E. coli 21,600 32 kDa 2 No 5 Fc
E. coli 50,460 40-45 40 45 kDa 6 No 5-8.2 582 Fc
E. coli 35,800 36 kDa 4 No 7.5 75 VL-kappa
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Pain: 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 结合F 区和轻链 对于IgA, IgM, IgE, 等相对结合力较弱
Protein P t i A/G
Protein 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 A
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A/G+ is the most robust IgG binding protien
A/G+
L FT PD
A+
FT PD
G+
Control
Key: L = Lysate Load FT = Flow Through PD = S Sample P ll Down l Pull D Control = Lysate + resin (no antibody)
FT PD FT PD
Anti-PP2A (Mouse) Anti-Cdk2 (Rabbit) ( ) Anti-p53 ( p (Sheep) p)
Conclusion: Thermo Scientific Pierce Classic IP Kit is compatible with most IgG species isotypes.
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