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@ARTICLE{Tashev:292129,
author = {S. A. Tashev and J. Euchner and K. Yserentant and S.
Hänselmann and F. Hild and W. Chmielewicz and J. Hummert
and F. Schwörer$^*$ and N. Tsopoulidis and S. Germer and Z.
Saßmannshausen and O. T. Fackler and U. Klingmüller$^*$
and D.-P. Herten},
title = {{P}ro{DOL}: a general method to determine the degree of
labeling for staining optimization and molecular counting.},
journal = {Nature methods},
volume = {21},
number = {9},
issn = {1548-7091},
address = {London [u.a.]},
publisher = {Nature Publishing Group},
reportid = {DKFZ-2024-01624},
pages = {1708-1715},
year = {2024},
note = {#LA:B200# / 2024 Sep;21(9):1708-1715},
abstract = {Determining the label to target ratio, also known as the
degree of labeling (DOL), is crucial for quantitative
fluorescence microscopy and a high DOL with minimal
unspecific labeling is beneficial for fluorescence
microscopy in general. Yet robust, versatile and easy-to-use
tools for measuring cell-specific labeling efficiencies are
not available. Here we present a DOL determination technique
named protein-tag DOL (ProDOL), which enables fast
quantification and optimization of protein-tag labeling.
With ProDOL various factors affecting labeling efficiency,
including substrate type, incubation time and concentration,
as well as sample fixation and cell type can be easily
assessed. We applied ProDOL to investigate how human
immunodeficiency virus-1 pathogenesis factor Nef modulates
CD4 T cell activation measuring total and activated copy
numbers of the adapter protein SLP-76 in signaling
microclusters. ProDOL proved to be a versatile and robust
tool for labeling calibration, enabling determination of
labeling efficiencies, optimization of strategies and
quantification of protein stoichiometry.},
cin = {B200},
ddc = {610},
cid = {I:(DE-He78)B200-20160331},
pnm = {312 - Funktionelle und strukturelle Genomforschung
(POF4-312)},
pid = {G:(DE-HGF)POF4-312},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:39117875},
doi = {10.1038/s41592-024-02376-6},
url = {https://inrepo02.dkfz.de/record/292129},
}