Method of determining extinction coefficient of fluorescent dye and protein concentration of dye-protein conjugate

摘要

The extinction coefficient of fluorescent dyes in donor- acceptor energy transfer processes is determined for dye-protein conjugates by forming short polypeptide fragment-dye conjugates and measuring the change in ultraviolet light absorbance of the conjugates with changing concentration. The protein concentration is determined by comparing the absorption spectrum of a conjugate of donor dye, acceptor dye, and protein with the individual components thereof using a multiple linear regression technique based on the following model:P PA.sub. p-d =&agr;·A.sub.p +&bgr;·A.sub.d +&egr; P P where, A.sub.p-d, A.sub.p, A.sub.d are the absorption spectra of the dye-protein conjugate, the protein alone, and the dye- polypeptide compound, respectively, &agr;, &bgr; are the regression coefficients to be determined, and &egr; is the error term.