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Methods Mol Biol. 2018;1649:85-94. doi: 10.1007/978-1-4939-7213-5_5.

Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo.

Author information

1
Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK.
2
Department of Anatomy, Brain Research Institute, and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.
3
Department of Anatomy, Brain Research Institute, and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea. hosungjung@yonsei.ac.kr.

Abstract

Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs. Here, we describe a detailed protocol to identify translated mRNAs in RGC axons in mouse in vivo. This method can be applied to any neurons whose cell bodies and distal axons are anatomically separated.

KEYWORDS:

Axon; Immunoprecipitation; Neuron; Ribosome; Translation; mRNAs

PMID:
29130191
DOI:
10.1007/978-1-4939-7213-5_5
[Indexed for MEDLINE]

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