由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析
Summary
细胞透水交联剂DSP [dithiobis（琥珀酰亚胺丙酸）]稳定短暂和不稳定的相互作用<em>在体内</em>，这使得他们的隔离，使用严格的蛋白质复合体的净化技术。在这里，我们目前培养的交联，由免疫蛋白复合物的分离细胞的技术。
Abstract
蜂窝机械的动态性质经常是建立在瞬态和/或弱蛋白协会。这些低亲和力相互作用排除围绕一个感兴趣的蛋白质，蛋白质网络的隔离和识别的严格方法。使用化学交联剂允许的不稳定相互作用的选择性稳定，从而绕过生化净化的局限性。在这里，我们提出在文化协议细胞适合使用一个homobifunctional交联剂间隔臂12埃，dithiobis（琥珀proprionate）（DSP）。 DSP是一个分子中的二硫键还原裂解。交联与磁珠的利益的蛋白质与免疫层析相结合，使蛋白质复合物，否则将不能承受净化隔离。此协议是与常规免疫印迹技术兼容，它可以扩展为蛋白质鉴定的质谱1。
斯蒂芬妮A. Zlatic和珠江五莱德贡献同样对这项工作。
Protocol
1。准备交联您将需要板足够数量的细胞，让500微克每标准管反应蛋白的隔离。单管可能足以确定一个由免疫印迹的兴趣的蛋白质公认interactors。在这种情况下，珠约束的材料可以用SDS – PAGE样品缓冲液（免疫）洗脱。标准反应质谱分析，应增加至少十倍，应使用的抗体（免疫亲和层析）认可的一种肽类抗原竞争洗脱的蛋白质复合物。这一战略将让免疫球蛋白的蛋白质复合物的隔离。 …
Discussion
DSP，膜渗透，化学还原与12 Å间隔臂的交联剂是用来稳定瞬态蛋白质相互作用 1,2,3,4 。在这里，我们体现这一战略与AP – 3适配器复杂的一种可溶性蛋白复合物，囊泡膜蛋白识别和排序，从内涵体 5 。 AP – 3的选择性结合的锌转运ZnT3和脂质激酶磷酸肌醇4激酶II型阿尔法但不转铁蛋白受体 1,4,6 。我们扩大了这些意见，以确定质谱 1膜蛋白和胞质因素AP – 3蛋白的相互作用…
Acknowledgements
这项工作是支持的美国国立卫生研究院的VF（NS42599和GM077569）的赠款。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Phosphate Buffered Saline | Invitrogen | P4417 | Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM | |
| Dithiobis (succinimidyl propionate) (DSP) | Thermo Scientific | 22585 | Moisture sensitive, store in air tight container 4°C | |
| Dimethyl sulphoxide (DMSO) Hybri-Max | Sigma | D2650 | ||
| Triton X-100, SigmaUltra | Sigma | T9284 | ||
| Dynabeads, Sheep anti-Mouse IgG | Invitrogen | 110.31 | Beads are also available as sheep anti-rabbit | |
| Dyna-Mag-2 magnet | Invitrogen | 123-21D |
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Tags
IsolationLabile Multi-protein ComplexesIn VivoControlled Cellular Cross-linkingImmuno-magnetic Affinity ChromatographyDynamic NatureTransient Protein AssociationsWeak Protein AssociationsLow Affinity InteractionsIsolation MethodsIdentification MethodsProtein NetworksChemical CrosslinkersSelective StabilizationBiochemical LimitationsPurification MethodsProtocolCells In CultureHomobifunctional CrosslinkerSpacer ArmDithiobis-(succinimidyl Proprionate)CleavageReductionDisulphide BondCross-linking And Immunoaffinity ChromatographyMagnetic BeadsProtein ComplexesPurification TechniquesWestern Blot TechniquesMass Spectrometry
Citer Cet Article
Zlatic, S. A., Ryder, P. V., Salazar, G., Faundez, V. Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography. J. Vis. Exp. (37), e1855, doi:10.3791/1855 (2010).