免疫共沉淀结合双向电泳分离相互作用蛋白质的方法优化

Establishment and Optimization of Co-immunoprecipitation Combined with Two-dimensional Electrophoresis

目的:建立和优化有针对性的免疫共沉淀(CO-IP)与双向电泳(2DE)-质谱联合的技术来鉴定相互作用蛋白质。方法:利用特异性抗体通过CO-IP对细胞全蛋白中的目标蛋白进行富集,用不同洗脱液洗脱目标及其相互作用蛋白,并行2DE分离。结果:对CO-IP过程、洗脱液成分、上样量等关键步骤进行优化,得到满意的2DE图谱。结论:成功优化CO-IP与2DE分离技术,为通过蛋白质组学研究信号通路中重要蛋白质的相互作用提供有效方法。

Objective： To establish and optimize the combination of targeted co-immuneprecipitation（CO-IP）, two-dimensional electrophoresis（2DE）and mass spectrometry technology for identification the fimction of protein. Methods： CO-IP was used to enrich and purify the proteins related to target proteins from total protein of the cells, and the 2DE was used to separate them with different elution solutions. Results： The key steps were optimized on the CO-IP process, loading amount of sample and elution composition and the best 2DE conditions were yielded. Conclusion： It achieves the quick separation and identification of the assosiated proteins of the key protein in the signal pathway, and also provides the effective way for identification of related proteins by proteomics.