Abstract
Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and “clickable” amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling by "click" chemistry. Here we describe a protocol to measure protein synthesis by AHA labeling and flow cytometry. Taking advantage of gating different cell populations, we provide a typical example of the flow cytometric-based analysis of protein synthesis during the cell cycle. While we used mouse B cells in this protocol this method can be readily applied to any cell types and organisms.
Keywords: Protein synthesis, Translation, AHA, Click chemistry, Flow cytometry, Cell cycle, Mitosis, Non-radioactive amino acid
Background
Traditionally measurement of protein synthesis is performed by pulse labeling of translation products using radiolabeled amino acids such as [35S]methionine and [35S]cysteine but the use of radioactive materials is the major disadvantage. Recent advance in bio-orthogonal chemical reporters such as azides and alkynes allows us to quantitatively monitor and track biomolecules (proteins, lipids or nucleic acids) through click chemistry (Dieterich, 2010). Azidohomoalaine (AHA) (Figure 1) is a methionine analog that contains an azide moiety and is incorporated into newly synthesized proteins. Thus, nascent proteins can be detected through "click" reaction between AHA (azido) and a fluorescent alkyne (Dieterich, 2010).Figure 1. Chemical structures of L-methionine and L-azidohomoalaine (AHA)Measuring protein synthesis during the cell cycle in mammalian cells has been challenging. To do this, cell synchronization in specific phases of the cell cycle is necessary but the use of drugs such as nocodazole was shown to affect translation (Coldwell et al., 2013; Shuda et al., 2015). To measure protein synthesis without drug treatment, here we used metabolic pulse labeling with AHA in a flow cytometric assay (Kiick et al., 2002; Shuda et al., 2015). In combination with AHA, we used anti-phospho-Histone H3 (Ser10) antibody as a mitotic marker to monitor mitotic translation in mouse B lymphoma cells (19DN) (Sander et al., 2015).
Materials and Reagents
Equipment
Software
Procedure
Recipes
Notes
You can perform washing, fixing, click reaction and etc. in a 96-well U- or V-bottom cell culture plate to increase the throughput. You can sort the AHA-labeled cells and the sorted cells can be used for many applications including western blot analysis and quantitative analysis using mass spectrometry.
Acknowledgments
We would like to thank Shuda et al. for describing the original protocol (Shuda et al., 2015). We adapted and modified the protocol for this study. We would also like to thank Matthias Selbach (Max Delbruck Center for Molecular Medicine) for critical reading of the manuscript and for his advice.
Competing interests
The authors declare no conflicts of interest or competing interests.
References
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