Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for measurements

Batta, Ágnes
Hajdu, Tímea
Nagy, Peter
Batta Ágnes (2023-) (xxx)
Folyóirat címe
Folyóirat ISSN
Kötet címe (évfolyam száma)
Forster resonance energy transfer (FRET) is a radiationless interaction between a donor and an acceptor whose distance dependence makes it a sensitive tool for studying the oligomerization and the structure of proteins. When FRET is determined by measuring the sensitized emission of the acceptor, a parameter characterizing the ratio of detection efficiencies of an excited acceptor versus an excited donor is invariably involved in the formalism. For FRET measurements involving fluorescent antibodies or other external labels, this parameter, designated by alpha, is usually determined by comparing the intensity of a known number of donors and acceptors in two independent samples leading to a large statistical variability if the sample size is small. Here, we present a method that improves precision by applying microbeads with a calibrated number of antibody binding sites and a donor-acceptor mixture in which donors and acceptors are present in a certain, experimentally determined ratio. A formalism is developed for determining alpha and the superior reproducibility of the proposed method compared to the conventional approach is demonstrated. Since the novel methodology does not require sophisticated calibration samples or special instrumentation, it can be widely applied for the quantification of FRET experiments in biological research.
Cytometry Part A. -103 : 7 (2023), p. 563-574. -Cytom. Part A. -1552-4922. -1552-4930