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  • Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo.

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo.

STAR protocols (2021-09-30)
Daisy J Vinter, Caroline Hoppe, Hilary L Ashe
ABSTRACT

Significant regulation of gene expression is mediated at the translation level. Here, we describe protocols for imaging and analysis of translation at single mRNA resolution in both fixed and living Drosophila embryos. These protocols use the SunTag system, in which the protein of interest is visualized by inserting a peptide array that is recognized by a single chain antibody. Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, Molecular Biology, ≥97.0%
Sigma-Aldrich
TWEEN® 20, Molecular Biology, viscous liquid
Sigma-Aldrich
Phosphate buffered saline, tablet
Sigma-Aldrich
Dextran sulfate sodium salt from Leuconostoc spp., Molecular Biology, average Mw >500,000 (dextran starting material), contains 0.5-2% phosphate buffer
Sigma-Aldrich
Triton X-100, laboratory grade