Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM.
Journal
Molecular biology of the cell
ISSN: 1939-4586
Titre abrégé: Mol Biol Cell
Pays: United States
ID NLM: 9201390
Informations de publication
Date de publication:
01 06 2019
01 06 2019
Historique:
pubmed:
11
4
2019
medline:
9
1
2020
entrez:
11
4
2019
Statut:
ppublish
Résumé
How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.
Identifiants
pubmed: 30969885
doi: 10.1091/mbc.E18-10-0661
pmc: PMC6724688
doi:
Substances chimiques
Luminescent Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1369-1376Références
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