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MethodsX. 2019 Apr 29;6:998-1008. doi: 10.1016/j.mex.2019.04.025. eCollection 2019.

Methods toward simplification of time resolved fluorescence anisotropy in proteins labeled with NBD (4-chloro-7-nitrobenzofurazan) adducts.

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Departments of Ophthalmology, Pathology and Laboratory Medicine, Jules Stein Eye Institute, University of California, 100 Stein Plaza Rm. BH 623, Los Angeles, CA 90095, United States.


The analysis of time resolved fluorescence anisotropy for NBD tagged proteins is difficult when multiple exponential components arise from heterogeneous amino acid fluorescent adducts. Two approaches were taken toward simplification. First, N terminal selective labeling of tear lipocalin with NBD-Cl was attempted at pH 7.0. While lysines were predominantly labeled at pH 8.0, selective N terminal labeling was attained at neutral pH. Second, fluorescence anisotropic decay analysis was simplified to recover only the rotational correlation time of the protein not the side chain. The boundaries for analysis of anisotropic decays were limited to the longer lifetimes. A modified tail fit enabled fitting the anisotropic decay to a single exponential. The correlation time for tear lipocalin matched published values. Additionally, a method for normalization of acquisition times of vertically (VV) and horizontally (VH) polarized fluorescence emission decays is presented for time-resolved anisotropy. Here it is applied to Picoharp software (Picoquant, Berlin). Picoharp software is programmed with an automatic stop at unequal acquisition times if the fluorescent counts exceeds a default. The method adjusts the intensity decays to the same acquisition time and is applicable to all time-resolved anisotropic decay data collected with time-correlated photon counting. •NBD labeling at pH 7.0 was not selective for N terminus of LCN1.•Constraints for range simplifies fittings of anisotropic decays.•Different acquisition times for decays can be normalized to facilitate fitting in data obtained by Picoharp.


Constrained tailfit for parameter reduction in time resolved fluorescence anisotropy; Fluorescence anisotropy; Fluorescence lifetime analysis; LCN1; Lipocalin 1; N terminal labeling of proteins; Normalization of VH; Time correlated single photon counting; pH selectivity of NBD-Cl

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