Comparison of Methods to Classify and Quantify Free and Bound States of Complexes using Single Molecule Fluorescence Anisotropy

Sean Burrows, Dimitrios Pappas

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Steady-state single molecule fluorescence anisotropy (SMFA) is described to quantify free and bound probe molecules from a Biotin-Neutravidin complexation reaction. By formulating a ratio of bound to total number of molecules sampled (Nb/Nt ratio) we quantified the extent of binding. We report on a comparison of three methods to extract fluorescent bursts from single molecules from a ten-minute time trace. The impact on the Nb/Nt ratio using either anisotropy values alone or anisotropy values combined with the difference in detector counts (∆n) were investigated. The data analysis methods reduced the random error due to scatter. Biotin-Rhodamine 110 (BR110) was used as the labeled probe for these studies. Neutravidin was used as the target protein. A competitive reaction between labeled BR110 probe and unlabeled Biotin was also investigated. The use of steady-state SMFA as a tool to probe molecular complexation will be useful in performing sensitive immunoassays, in drug disco
Original languageEnglish
Pages (from-to)1911-1921
JournalThe Analyst
StatePublished - 2009


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