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

Ullrich, Milena, Liang, Vanessa, Chew, Yee Lian, Banister, Samuel, Song, Xiaomin, Zaw, Thiri, Lam, Hong, Berber, Slavica, Kassiou, Michael, Nicholas, Hannah R. and Goetz, Juergen (2014) Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans. Nature Protocols, 9 9: 2237-2255. doi:10.1038/nprot.2014.150


Author Ullrich, Milena
Liang, Vanessa
Chew, Yee Lian
Banister, Samuel
Song, Xiaomin
Zaw, Thiri
Lam, Hong
Berber, Slavica
Kassiou, Michael
Nicholas, Hannah R.
Goetz, Juergen
Title Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans
Journal name Nature Protocols   Check publisher's open access policy
ISSN 1750-2799
1754-2189
Publication date 2014-09-01
Year available 2014
Sub-type Critical review of research, literature review, critical commentary
DOI 10.1038/nprot.2014.150
Volume 9
Issue 9
Start page 2237
End page 2255
Total pages 19
Place of publication London, United Kingdom
Publisher Nature Publishing Group
Collection year 2015
Language eng
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.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Critical review of research, literature review, critical commentary
Collections: Queensland Brain Institute Publications
Official 2015 Collection
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 12 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 11 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Sun, 16 Nov 2014, 11:16:39 EST by System User on behalf of Queensland Brain Institute