In:
Molecular Biology of the Cell, American Society for Cell Biology (ASCB), Vol. 28, No. 21 ( 2017-10-15), p. 2747-2756
Abstract:
Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging.
Type of Medium:
Online Resource
ISSN:
1059-1524
,
1939-4586
DOI:
10.1091/mbc.e17-03-0161
Language:
English
Publisher:
American Society for Cell Biology (ASCB)
Publication Date:
2017
detail.hit.zdb_id:
1474922-1
SSG:
12
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