Unit

UNIT 7.11 Metabolic Labeling with Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging

  1. Susanne tom Dieck1,
  2. Anke Müller2,3,
  3. Anne Nehring1,
  4. Flora I. Hinz1,
  5. Ina Bartnik1,
  6. Erin M. Schuman1,
  7. Daniela C. Dieterich2,3

Published Online: 1 SEP 2012

DOI: 10.1002/0471143030.cb0711s56

Current Protocols in Cell Biology

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.

Author Information

  1. 1

    Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Frankfurt, Germany

  2. 2

    Leibniz Institute for Neurobiology, Research Group Neuralomics, Magdeburg, Germany

  3. 3

    Otto-von-Guericke-University Magdeburg, Medical Faculty, Institute for Pharmacology and Toxicology, Magdeburg, Germany

Publication History

  1. Published Online: 1 SEP 2012
  2. Published Print: SEP 2012

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.

Keywords:

  • FUNCAT;
  • click chemistry;
  • copper(I)-catalyzed [3+2]azide-alkyne cycloaddition;
  • AHA;
  • HPG;
  • protein synthesis