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

Hinz, Flora I. and Dieterich, Daniela C. and Tirrell, David A. and Schuman, Erin M. (2012) Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish. ACS Chemical Neuroscience, 3 (1). pp. 40-49. ISSN 1948-7193 . http://resolver.caltech.edu/CaltechAUTHORS:20120214-110853304

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/cn2000876 DOIArticle
http://pubs.acs.org/doi/abs/10.1021/cn2000876PublisherArticle
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
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.
Funders:
Funding AgencyGrant Number
Max-Planck SocietyUNSPECIFIED
NIHGM62523
NIH/NRSA Institutional training grant5T32 GM07616
Record Number:CaltechAUTHORS:20120214-110853304
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120214-110853304
Official Citation:Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish Flora I. Hinz, Daniela C. Dieterich, David A. Tirrell, and Erin M. Schuman ACS Chemical Neuroscience20123 (1), 40-49
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:29277
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Mar 2012 20:29
Last Modified:19 Dec 2014 23:42

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