Valdez-Sinon Arielle Nicole, Gokhale Avanti, Faundez Victor, Bassell Gary Jonathan
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
STAR Protoc. 2020 Aug 11;1(2):100083. doi: 10.1016/j.xpro.2020.100083. eCollection 2020 Sep 18.
This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive immunoprecipitation and FLAG peptides permit efficient elution of protein complexes. With many commercially available FLAG tools, this protocol is highly versatile. This procedure reduces immunoprecipitation of nonspecific binding proteins. Gene ontology analyses performed following mass spectrometry-based proteomics may elucidate novel functions of proteins of interest. For complete details on the use and application of this protocol, please refer to Valdez-Sinon et al. (2020).
本方案描述了与FLAG标签重组蛋白相关的蛋白质的免疫沉淀，随后进行基于质谱的蛋白质组学以鉴定相关的相互作用组成分。选择FLAG表位是因为现有的高亲和力单克隆抗体可实现灵敏的免疫沉淀，且FLAG肽可有效洗脱蛋白质复合物。有许多市售的FLAG工具，本方案具有高度通用性。该程序减少了非特异性结合蛋白的免疫沉淀。基于质谱的蛋白质组学之后进行的基因本体分析可能会阐明感兴趣蛋白质的新功能。有关本方案使用和应用的完整详细信息，请参考瓦尔迪兹 - 西农等人（2020年）的文献。
