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. 2008 Jul;25(7):1487-99.
doi: 10.1007/s11095-007-9516-9. Epub 2008 Jan 3.

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Free PMC article

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Andrea Hawe et al. Pharm Res. .
Free PMC article


Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.


Fig. 1
Fig. 1
Simplified scheme showing the energy levels of the ground state So and various excited states, as well as electronic transitions taking place for fluorescent dyes. After light absorption, where the electrons are lifted from the ground state S0 to higher energetic excited levels (bold dashed dotted arrow; for sake of clarity, absorption to only one vibrational level of the S2 excited state level is shown), radiationless vibrational relaxation and internal conversion processes (dashed arrows), solvent relaxation and (twisted) intramolecular charge transfer (T)ICT (dotted arrows), as well as fluorescence (solid arrows) can occur. For sake of clarity the conversion to the triplet state, which can occur from S(T)ICT is not included in this figure.
Fig. 2
Fig. 2
Chemical structures of commonly used fluorescent dyes: ANS (A), Bis-ANS (B), Nile Red (C), DCVJ (D), Thioflavin T (E) and Congo Red (F).
Fig. 3
Fig. 3
Polarity dependent fluorescence emission of 1 μM ANS excited at 350 nm (A), 1 μM Bis-ANS excited at 385 nm (B) and 1 μM Nile Red excited at 550 nm (C) in different solvents.

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