Elion Elaine A
Harvard Medical School, Boston, Massachusetts, USA.
Curr Protoc Neurosci. 2006 May;Chapter 5:Unit 5.25. doi: 10.1002/0471142301.ns0525s35.
Coprecipitation of proteins from whole-cell extracts is a valuable approach to testing for physical interactions between proteins of interest. When a precipitating antibody is used, this method is referred to as co-immunoprecipitation. Coprecipitation can be used to study interactions between known proteins and as a means of identifying components of a complex. This unit describes basic approaches to immunoprecipitating tagged proteins from whole-cell extracts. The extract is prepared under nondenaturing conditions, the protein of interest is precipitated, and the precipitate is tested for a second specifically associated protein. The approach can be used for native or epitope-tagged proteins for which antibodies are available, or for recombinant proteins that bind with high affinity to a molecule that can be coupled to a solid-phase matrix. An associated protein is detected by separating the precipitated proteins by SDS-PAGE and immunoblotting with a second antibody that recognizes the putative associated protein.
从全细胞提取物中共沉淀蛋白质是检测目标蛋白质之间物理相互作用的一种有价值的方法。当使用沉淀抗体时，这种方法被称为免疫共沉淀。共沉淀可用于研究已知蛋白质之间的相互作用，并作为鉴定复合物成分的一种手段。本单元描述了从全细胞提取物中免疫沉淀标记蛋白质的基本方法。提取物在非变性条件下制备，目标蛋白质被沉淀，然后对沉淀物检测是否存在第二种特异性相关蛋白质。该方法可用于有抗体可用的天然蛋白质或表位标记蛋白质，或用于与可偶联到固相基质的分子具有高亲和力结合的重组蛋白质。通过SDS-PAGE分离沉淀的蛋白质并用识别假定相关蛋白质的第二种抗体进行免疫印迹来检测相关蛋白质。
