Identification of Nanchangmycin protein targets

HeLa cells were treated with Nan-Dyne, a nanchangmycin probe bearing both alkyne and diazirine, irradiated with UV. For competition experiments, cells were also treated with an excess of nanchangmycin. Cells were irradiated with UV, lysed, clicked with azido-biotin, and proteins enriched on neutravidin beads. Samples were eluted on a SDS-Page gel and in-gel digested with trypsin.

将海拉细胞（HeLa cells）经携带炔基（alkyne）与双吖丙啶（diazirine）的南昌霉素探针Nan-Dyne处理后，施以紫外线（UV）照射。针对竞争性实验，细胞需同时用过量南昌霉素进行处理；随后所有组别细胞均进行裂解、采用叠氮生物素（azido-biotin）完成点击化学反应，并通过中性亲和素磁珠（neutravidin beads）富集靶蛋白。将样品在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）胶上进行洗脱后，以胰蛋白酶（trypsin）进行胶内酶解。