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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Change history
07 September 2017
In the version of this article initially published, there was an error in Figure 1b. The structure of triazole, a 5-membered ring structure generated after the click reaction, was mistakenly drawn as a 6-membered ring. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
This work was supported in part by the National Research Foundation-supported Interdisciplinary Research Group in Infectious Diseases of SMART (Singapore-MIT Alliance for Research and Technology) and by research grants from the National Medical Research Council Singapore (nos. NMRC-CIRG/1346/2012 and NMRC/CIRG/1373/2013) to H.-M.S. Y.M.L. was supported by NUS Research Scholarships. We also acknowledge support from the Chinese National Natural Sciences Foundation (grant nos. 81630092 and 81421091) and the Doctoral Station Science Foundation from the Chinese Ministry of Education of China (grant no. 20130091130003 to Z.-C.H.).
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J.W., J.Z., Q.L. and H.-M.S. designed the research. J.W. and J.Z. performed the experiments. Z.-C.H., S.N. and Y.S. assisted in the data analysis. J.W., J.Z., Y.M.L., Q.L. and H.-M.S. wrote the manuscript.
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Wang, J., Zhang, J., Lee, Y. et al. Nonradioactive quantification of autophagic protein degradation with L-azidohomoalanine labeling. Nat Protoc 12, 279–288 (2017). https://doi.org/10.1038/nprot.2016.160
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DOI: https://doi.org/10.1038/nprot.2016.160
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