串联亲和纯化
免疫沉淀
表位
标志标签
化学
抗体
分子生物学
串联质谱法
亲和层析
单克隆抗体
生物化学
色谱法
生物
重组DNA
融合蛋白
质谱法
酶
基因
免疫学
作者
James A. DeCaprio,Thomas Kohl
出处
期刊:CSH Protocols
[Cold Spring Harbor Laboratory]
日期:2019-02-01
卷期号:2019 (2): pdb.prot098657-pdb.prot098657
被引量:8
标识
DOI:10.1101/pdb.prot098657
摘要
The immunoaffinity purification of target proteins followed by the identification and characterization of associated proteins by mass spectrometry is a widely used technique. An immunoaffinity purification bears resemblance to a standard immunoprecipitation; however, the end product for mass spectrometric analysis in the femtomole (10 −15 ) to attomole (10 −18 ) range is required to be of exceptional purity. This high degree of sensitivity in detection renders it of extreme importance to eliminate most if not all of the nonspecific background proteins and can be achieved by performing a tandem affinity purification (TAP). In TAP, the cDNA of the target protein is engineered to contain at least two different epitope tags, and the target protein is extracted under nondenaturing conditions upon expression using an appropriate protein expression platform (CHO cells, HEK 293 cells, or yeast). The expressed protein is initially immunoprecipitated using an antibody against one epitope tag and is eluted in the presence of excess peptide by competition for antibody-binding sites, before being reimmunoprecipitated using an antibody that specifically recognizes the second epitope. These sequential immunoprecipitations significantly reduce the presence of associated nonspecific proteins. Numerous combinations of epitope tags have been applied for tandem affinity purification, and this protocol illustrates the use of tandem hemagglutinin (HA) and FLAG epitope tags. The first immunoprecipitation uses an anti-FLAG antibody followed by the elution in the presence of a competing FLAG peptide before the reimmunoprecipitation of the protein using an anti-HA antibody. Numerous high-quality antiepitope tag antibodies are commercially available from different antibody manufacturers.
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