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Nat Protoc. 2014 Sep;9(9):2237-55. doi: 10.1038/nprot.2014.150. Epub 2014 Aug 28.

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

Author information

1
1] Brain and Mind Research Institute, University of Sydney, Camperdown, New South Wales, Australia. [2] Institute for Integrative Neuroanatomy, Charité, Universitätsmedizin Berlin, Berlin, Germany.
2
Brain and Mind Research Institute, University of Sydney, Camperdown, New South Wales, Australia.
3
School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia.
4
School of Chemistry, University of Sydney, Camperdown, New South Wales, Australia.
5
Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales, Australia.
6
1] School of Chemistry, University of Sydney, Camperdown, New South Wales, Australia. [2] Faculty of Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
7
1] School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia. [2].
8
1] Brain and Mind Research Institute, University of Sydney, Camperdown, New South Wales, Australia. [2] Clem Jones Centre for Ageing Dementia Research (CJCADR), Queensland Brain Institute (QBI), The University of Queensland, Brisbane, Queensland, Australia. [3].

Erratum in

  • Nat Protoc. 2014 Dec;9(12):2903.

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.

PMID:
25167056
DOI:
10.1038/nprot.2014.150
[Indexed for MEDLINE]
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