Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans

Milena Ullrich, Vanessa Liang, Jürgen Götz, Yee Lian Chew, Samuel Banister, Xiaoming Song, Thiri Zaw, Hong Lam, Slavica Berber, Michael Kassiou, Hannah R. Nicholas

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo-synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo-synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1-2 d for bio-orthogonal labeling; and, with regard to analysis, 3-4 d for western blotting, 5-6 d for immunofluorescence or ~3 weeks for mass spectrometry.
Original languageEnglish
Pages (from-to)2237-2255
Number of pages19
JournalNature Protocols
Volume9
Issue number9
DOIs
Publication statusPublished - Sep 2014

Bibliographical note

A corrigendum exists for this article and can be found in Nature Protocols, 9, 2903, doi: 10.1038/nprot1214-2903c

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