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Nonradioactive quantification of autophagic protein degradation with L-azidohomoalanine labeling

Wang, Jigang, Zhang, Jianbin, Lee, Yew Mun, Ng, Shukie, Shi, Yin, Hua, Zi-Chun, Lin, Qingsong, Shen, Han-Ming·2017·Nature Protocols
DOI 10.1038/nprot.2016.160
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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.

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Metadata

Authors
Wang, J., Zhang, J., Lee, Y. M., Ng, S., Shi, Y., Hua, Z.-C., Lin, Q., & Shen, H.-M.
ORCID
Venue
Nature Protocols
Publisher
Springer Science and Business Media LLC
Published
2017-01-12
Volume
12
Issue
2
Pages
279-288
ISSN
  • 1754-2189
  • 1750-2799
DOI
10.1038/nprot.2016.160
Cited by
52
References
7
Language
en
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