Nonradioactive quantification of autophagic protein degradation with L-azidohomoalanine labeling.
作者： Jigang Wang , Jianbin Zhang , Yew Mun Lee , Shukie Ng , Yin Shi
关键词:
摘要: At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) protein during autophagy. AHA is used as surrogate for L-methionine, and, when added cultured cells grown in methionine-free medium, incorporated into de novo synthesis. After chase period remove short-lived proteins, autophagy induced by starvation or other stimuli. Cells then undergo 'click' reaction between azide group and fluorescently tagged alkyne probe. The AHA-containing can be detected flow cytometry. This nonradioactive, sensitive quantitative, it easy perform. It also applicable various cell culture systems. whole estimated take 4-5 d complete.
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