Quantitative determination of fluorescence labeling implemented in cell cultures

BMC Biology

Methodology Article

Chiara Schirripa Spagnolo¹  Aldo Moscardini¹  Fabio Beltram²  Stefano Luin²  Rosy Amodeo³ 

[1]NEST Laboratory, Scuola Normale Superiore, Piazza San Silvestro 12, 56127, Pisa, Italy

[2]NEST Laboratory, Scuola Normale Superiore, Piazza San Silvestro 12, 56127, Pisa, Italy

[3]NEST Laboratory, Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127, Pisa, Italy

[4]NEST Laboratory, Scuola Normale Superiore, Piazza San Silvestro 12, 56127, Pisa, Italy

[5]Present address: Department of Biomedical Sciences, Humanitas University, Via Rita Levi Montalcini 4, Pieve Emanuele, 20072, Milan, Italy

关键词: Fluorescent labeling;    Fluorescence microscopy;    Single-molecule imaging;    Single-particle tracking;    Membrane receptors;    Sfp phosphopantetheinyl transferase;   

DOI  :  10.1186/s12915-023-01685-0

 received in 2022-11-29, accepted in 2023-08-18,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundLabeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative.ResultsWe present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, and reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4′-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling.ConclusionsThe developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions.

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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