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Published January 2012 | Supplemental Material
Journal Article Open

Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish

Abstract

Protein expression in the nervous system undergoes regulated changes in response to changes in behavioral states, in particular long-term memory formation. Recently, methods have been developed (BONCAT and FUNCAT), which introduce noncanonical amino acids bearing small bio-orthogonal functional groups into proteins using the cells' own translational machinery. Using the selective "click reaction", this allows for the identification and visualization of newly synthesized proteins in vitro. Here we demonstrate that noncanonical amino acid labeling can be achieved in vivo in an intact organism capable of simple learning behavior, the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, stimulation with a GABA antagonist that elicits seizures in the larval zebrafish causes an increase in protein synthesis throughout the proteome, which can also be visualized in intact larvae.

Additional Information

© 2011 American Chemical Society. Received: September 21, 2011; Accepted: November 7, 2011; Published: November 7, 2011. This work was supported by the Max-Planck Society. D.A.T. acknowledges support from NIH (GM62523). F.I.H. acknowledges support from NIH/NRSA Institutional training grant 5T32 GM07616. The authors would like to thank Mark Aizenberg for his help with behavioral tests, Georgi Tushev for his help with calculations of zebrafish proteome methionine content, Jennifer Hodas and John Ngo for general discussions and Stefanie Bunse and Susanne tom Dieck for comments on the manuscript.

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