Visualizing patterns of protein uptake to porous media using confocal scanning laser microscopy
Visualizing patterns of protein uptake to porous media using confocal scanning laser microscopy
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DOI：
10.1081/ss-120000319
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发表时间：
2002-01-01
影响因子：
2.8
通讯作者：
Thömmes, J
中科院分区：
文献类型：
--
作者：
Linden, T;Ljunglöf, A;Thömmes, J
共聚焦扫描激光显微镜已被用来可视化的摄取荧光标记的蛋白质多孔固定相在有限浴吸附实验。用三种不同的荧光染料标记参比蛋白质，并将多孔阳离子交换剂依次与这些蛋白质-染料缀合物的溶液孵育。该连续孵育实验用于研究吸附过程中蛋白质摄取的模式。在实验过程中获得的共聚焦图像清楚地显示，在某些条件下，吸附剂颗粒从边缘到核心逐渐饱和，这与收缩核心模型一致。移动的相条件（离子强度、pH）的变化可导致摄取模式的显著变化，即，将最初结合的分子进一步输送到核心，从而使边缘处的结合位点可用于吸附新分子。在低pH值和离子强度BSA的摄取模式，这是完全按照收缩核心模型，但从这个配置文件中观察到一个明显的偏差，在增加离子强度。在单克隆抗体的吸附过程中，pH值的变化足以完全改变有限浴吸附中的摄取模式。试图将这种行为与平衡结合等温线的形状相关联。这里提出的技术允许独特的见解蛋白质吸附到多孔介质的细节，并将提供一个有价值的扩展现有的实验方法的研究蛋白质色谱的机械方面。
Confocal scanning laser microscopy has been used to visualize the uptake of fluorescence-labeled proteins to porous stationary phases in finite bath adsorption experiments. Reference proteins were labeled with three different fluorescent dyes and a porous cation exchanger was sequentially incubated with solutions of these protein-dye conjugates. This sequential incubation experiment was used to investigate the pattern of protein uptake during adsorption. The confocal images obtained during the experiments clearly visualized that under certain conditions the adsorbent particles are gradually saturated from the rim to the core, a pattern consistent with the shrinking core model. Changes in the mobile phase conditions (ionic strength, pH) can lead to significant shifts in the uptake pattern, i.e., the further transport of initially bound molecules to the core, thus making binding sites at the rim available for adsorption of new molecules. At low pH and ionic strength BSA showed an uptake pattern, which was completely in accordance with the shrinking core model, but a clear deviation from this profile was observed at increased ionic strength. During adsorption of a monoclonal antibody a change in pH was sufficient to change the uptake pattern in finite bath adsorption completely. An attempt was made to correlate this behavior to the shape of the equilibrium-binding isotherm. The technique presented here allows unique insights into the details of protein adsorption to porous media and will provide a valuable extension of the existing experimental methods for studies of mechanistic aspects of protein chromatography.