Susanto Arthur, Herrmann Tim, Hubbuch Jürgen
Bioseparation Group, Institute of Biotechnology-2, Research Centre Jülich, 52425 Jülich, Germany.
J Chromatogr A. 2006 Dec 8;1136(1):29-38. doi: 10.1016/j.chroma.2006.09.053. Epub 2006 Oct 31.
Based on Lambert-Beer law and the light attenuation model, a new method will be introduced in this work in order to eliminate the disturbances caused by signal attenuation in the experimental data measured by confocal laser scanning microscopy (CLSM). This new method considers the attenuation effects which depend on concentration of fluorophore-labelled protein as well as attenuation effects which are independent from protein concentration. Furthermore, no solvent additive is required in order to match the refraction index of solvent to bead material. The determination of correction factors is, thus, easily done using the currently investigated chromatographic phase system, so that the validity of the correction factors in the current system can be guaranteed. The introduced correction method has been applied for the investigation of intraparticle protein distribution inside an HIC (hydrophobic interaction chromatography) particle.
基于朗伯-比尔定律和光衰减模型，本文将介绍一种新方法，以消除共聚焦激光扫描显微镜（CLSM）测量的实验数据中信号衰减所引起的干扰。这种新方法考虑了取决于荧光团标记蛋白浓度的衰减效应以及与蛋白浓度无关的衰减效应。此外，无需添加溶剂添加剂来使溶剂与珠子材料的折射率相匹配。因此，使用当前研究的色谱相系统很容易确定校正因子，从而可以保证当前系统中校正因子的有效性。所引入的校正方法已应用于研究疏水相互作用色谱（HIC）颗粒内部的颗粒内蛋白质分布。
