UNIT 7.11 Metabolic Labeling with Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging
Published Online: 1 SEP 2012
Copyright © 2012 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
tom Dieck, S., Müller, A., Nehring, A., Hinz, F. I., Bartnik, I., Schuman, E. M. and Dieterich, D. C. 2012. Metabolic Labeling with Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging. Current Protocols in Cell Biology. 56:7.11:7.11.1–7.11.29.
- Published Online: 1 SEP 2012
- Published Print: SEP 2012
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.
- click chemistry;
- copper(I)-catalyzed [3+2]azide-alkyne cycloaddition;
- protein synthesis