Determination of the Labeling Density of Fluorophore-Biomolecule Conjugates with Absorption Spectroscopy
Determination of the Labeling Density of Fluorophore-Biomolecule Conjugates with Absorption Spectroscopy
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DOI：
10.1021/bc2003428
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发表时间：
2012-02-01
影响因子：
4.7
通讯作者：
Resch-Genger, Ute
中科院分区：
文献类型：
--
作者：
Grabolle, Markus;Brehm, Robert;Resch-Genger, Ute
染料-生物分子的结合经常伴随着染料吸收光谱的显著变化，这限制了使用普通的光度方法来确定标记密度。在这里，我们描述了一种改进的方法，使用染料的积分吸光度而不是它在长波最大处的吸光度来确定生物分子偶联染料的浓度。以三种不同的菁染料为例说明了这一方法。
Dye-biomolecule conjugation is frequently accompanied by considerable spectral changes of the dye's absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.