title: Nonradioactive quantification of autophagic protein degradation with L-azidohomoalanine labeling.
abstract: At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli. Cells then undergo a 'click' reaction between the azide group of AHA and a fluorescently tagged alkyne probe. The AHA-containing proteins can then be detected by flow cytometry. This protocol is nonradioactive, sensitive and quantitative, and it is easy to perform. It is also applicable to various cell culture systems. The whole protocol is estimated to take 4-5 d to complete.
authors.affiliation: Changzhou High-Tech Research Institute of Nanjing University and the State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, China.|Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Jigang
authors.identifier: http://orcid.org/0000-0002-0575-0105
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Jianbin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Yew Mun
authors.identifier: http://orcid.org/0000-0002-8360-7689
authors.initials: YM
authors.lastname: Lee
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Shukie
authors.initials: S
authors.lastname: Ng
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Yin
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Changzhou High-Tech Research Institute of Nanjing University and the State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, China.
authors.forename: Zi-Chun
authors.initials: ZC
authors.lastname: Hua
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Qingsong
authors.initials: Q
authors.lastname: Lin
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.|NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore.
authors.forename: Han-Ming
authors.initials: HM
authors.lastname: Shen
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000480:Alkynes / Q000737:chemistry; D000818:Animals; D001343:Autophagy; D001386:Azides / Q000737:chemistry; D002460:Cell Line; D057930:Click Chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D051379:Mice; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D059748:Proteolysis; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000480:Alkynes; D001386:Azides; D005456:Fluorescent Dyes; D011506:Proteins; C506098:azidohomoalanine; D000409:Alanine