In:
Current Protocols in Cell Biology, Wiley, Vol. 56, No. 1 ( 2012-09)
Abstract:
Fluorescent labeling of proteins by genetically encoded fluorescent protein tags has enabled an enhanced understanding of cell biological processes but is restricted to the analysis of a limited number of identified proteins. This approach does not permit, e.g., the unbiased visualization of a full proteome in situ. We describe here a fluorescence‐based method to follow proteome‐wide patterns of newly synthesized proteins in cultured cells, tissue slices, and a whole organism. This technique is compatible with immunohistochemistry and in situ hybridization. Key to this method is the introduction of a small bio‐orthogonal reactive group by metabolic labeling. This is accomplished by replacing the amino acid methionine by the azide‐bearing methionine surrogate azidohomoalanine (AHA) in a step very similar to classical radioisotope labeling. Subsequently, an alkyne‐bearing fluorophore is covalently attached to the group by “click chemistry”—a copper(I)‐catalyzed [3+2]azide‐alkyne cycloaddition. By similar means, metabolic labeling can also be performed with the alkyne‐bearing homopropargylglycine (HPG) and clicked to an azide‐functionalized fluorophore. Curr. Protoc. Cell Biol . 56:7.11.1‐7.11.29. © 2012 by John Wiley & Sons, Inc.
Type of Medium:
Online Resource
ISSN:
1934-2500
,
1934-2616
DOI:
10.1002/0471143030.2012.56.issue-1
DOI:
10.1002/0471143030.cb0711s56
Language:
English
Publisher:
Wiley
Publication Date:
2012
detail.hit.zdb_id:
2179048-6
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