A rapid method for the determination of molar ratio of fluorochrome to protein techniques by fluorescence anisotropy detection
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The Determination of molar ratio of fluorochrome to protein is an important part in fluorescent antibody techniques. The conventional method is time consuming and with troublesome manipulations. A rapid homogeneous method based on the anisotropy change of the fluorochrome after reacting with protein was presented here. In our experiments, fluorescein isothiocyanate (FITC) and bovine serum albumin(BSA) were chosen to form the FITC/BSA conjugate. A series of solutions containing the two components were prepared and allow to react according to the standard procedure. Fluorescence anisotropy of the mixtures were detected, a diagram of r-lgc is then obtained, where c is the concentration of protein. Because FITC in the mixture exists in both free form(F) and binding form(B), the fluorescence anisotropy observed is given by r = f(F)r(F) + f(B)r(B), where r(F) and r(B) refer to the anisotropy of free and bound FITC, respectively; and f(F) and f(B) represent the fraction of the free and bound forms, respectively, f(F) + f(B) = 1. A formula as f(B) = (r-r(F))/(r(B)-r(F)) is achieved. Here r(B) correspods to the value of r in the upper platform region of the r-lgc curve, where FITC is bound to protein completely, while r(F) can be obtained by the determination of anisotropy of FITC in the absence of BSA. So, for each of the mixtures, the binding fraction of FITC can then be calculated. Correspondingly, the content of bound form of FITC that was used to calculate the molar ratio of FITC to BSA can be gained. Since the method avoids the tiresome separation procedure, it bears the merit of time saving and can be used to estimate the molar ratio of fluorochrome to protein rapidly. The determination results of samples by this method were compared with that got from a spectrophotometric analysis. The results were in good agreement.