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000292129 1001_ $$aTashev, Stanimir Asenov$$b0
000292129 245__ $$aProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting.
000292129 260__ $$aLondon [u.a.]$$bNature Publishing Group$$c2024
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000292129 520__ $$aDetermining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.
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000292129 7001_ $$aEuchner, Jonas$$b1
000292129 7001_ $$00000-0001-9746-6912$$aYserentant, Klaus$$b2
000292129 7001_ $$00000-0001-9006-6625$$aHänselmann, Siegfried$$b3
000292129 7001_ $$aHild, Felix$$b4
000292129 7001_ $$aChmielewicz, Wioleta$$b5
000292129 7001_ $$aHummert, Johan$$b6
000292129 7001_ $$00000-0003-4138-1412$$aSchwörer, Florian$$b7
000292129 7001_ $$00000-0002-8819-4350$$aTsopoulidis, Nikolaos$$b8
000292129 7001_ $$aGermer, Stefan$$b9
000292129 7001_ $$aSaßmannshausen, Zoe$$b10
000292129 7001_ $$00000-0003-2982-4209$$aFackler, Oliver T$$b11
000292129 7001_ $$0P:(DE-He78)860df4ab16c373fb28a815dcd81107a6$$aKlingmüller, Ursula$$b12$$eLast author$$udkfz
000292129 7001_ $$00000-0002-3884-0262$$aHerten, Dirk-Peter$$b13
000292129 773__ $$0PERI:(DE-600)2163081-1$$a10.1038/s41592-024-02376-6$$n9$$p1708-1715$$tNature methods$$v21$$x1548-7091$$y2024
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