TY - JOUR
T1 - ProDOL
T2 - A general method to determine the degree of labelling for staining optimisation and molecular counting
AU - Asenov Tashev, Stan
AU - Euchner, Jonas
AU - Yserentant, Klaus
AU - Hapfelmeier, Siegfried
AU - Hild, Felix
AU - Chmielewicz, Wioleta
AU - Hummert, Johan
AU - Schwörer, Florian
AU - Tsopoulidis, Nikolaos
AU - Germer, Stefan
AU - Saßmannshausen, Zoe
AU - Fackler, Oliver T.
AU - Klingmüller, Ursula
AU - Herten, Dirk
PY - 2024/3/26
Y1 - 2024/3/26
N2 - Determining the label to target ratio, also known as degree of labelling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labelling is beneficial for fluorescence microscopy in general. Yet, robust, versatile, and easy-to-use tools for measuring cell-specific labelling efficiencies are not available. This study presents a novel DOL determination technique named Protein-tag DOL (ProDOL), which enables fast DOL measurements and optimisation of protein-tag labelling. With ProDOL various factors affecting labelling 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 HIV-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adaptor protein SLP-76 in signalling microclusters. ProDOL proved to be a versatile and robust tool for labelling calibration, enabling determination of labelling efficiencies, optimisation of strategies, and quantification of protein stoichiometry.
AB - Determining the label to target ratio, also known as degree of labelling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labelling is beneficial for fluorescence microscopy in general. Yet, robust, versatile, and easy-to-use tools for measuring cell-specific labelling efficiencies are not available. This study presents a novel DOL determination technique named Protein-tag DOL (ProDOL), which enables fast DOL measurements and optimisation of protein-tag labelling. With ProDOL various factors affecting labelling 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 HIV-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adaptor protein SLP-76 in signalling microclusters. ProDOL proved to be a versatile and robust tool for labelling calibration, enabling determination of labelling efficiencies, optimisation of strategies, and quantification of protein stoichiometry.
UR - http://www.nature.com/nmeth/
M3 - Article
SN - 1548-7091
JO - Nature Methods
JF - Nature Methods
ER -
