A rapid method for the determination of molar ratio of fluorophore to protein by fluorescence anisotropy detection
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The determination of the fluorophore to protein molar ratio is an important part of fluorescent antibody techniques. A rapid and homogeneous method for the determination of fluorophore to protein molar ratio based on the fluorescence anisotropy variation of the fluorophore after reacting with protein is presented. In the work, fluorescein isothiocyanate (FITC) and bovine serum albumin (BSA) were chosen to form a FITC/BSA conjugate. In a series of reacting mixture solutions, the concentration of FITC was kept constant, while the concentration of BSA was varied, and the fluorescence anisotropies of the reacting mixture solutions were measured. By plotting the fluorescence anisotropy of FITC versus the logarithm value of the concentration of BSA, an important parameter which refers to the fluoresecence anisotropy when the fluorophore in the system was bound completely by protein, can be obtained. Combined with another parameter, fluorescence anisotropy in the absence of protein, the fraction of bound fluorophore in the samples can be achieved, and then the molar ratio of flurochrome to protein can be calculated. This method has been employed to the analysis of real samples, and the results well agree with that obtained by traditional method, indicating that this method is not only simple and rapid but also reliable, and can be possibly applied in practice. (C) 1999 Elsevier Science B.V. All rights reserved.