Direct coupling of immobilized metal ion affinity chromatography and capillary isoelectric focusing in a single capillary

K. Shimura, T. Nagai
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引用次数: 3

Abstract

A procedure for the direct coupling of affinity chromatography and capillary isoelectric focusing in a single capillary is described. A unified capillary device was made to implement this procedure. The inner surface of a fused-silica capillary was tandemly coated with a chelating polymer, polyiminodiacetate, at the inlet side and with a neutral polymer, polydimethylacrylamide, at the outlet side. After loading a fluorescence-labeled recombinant Fab with a hexahistidine tag, a model sample, the device was rinsed with a high-salt buffer and then with a carrier ampholyte solution to fill the device. The bound Fab was eluted by filling the nickel-chelate column with an anode solution, 100 mM phosphoric acid. A positive voltage was applied at the chelate column side with a pressure at the same side, so as to slightly overwhelm the anodic electroosmosis produced in the acidified chelate column and gradually move the focused protein bands through a stationary fluorescence detector. The mixture of the labeled recombinant Fab at variable concentration and labeled bovine serum albumin at 50 nM was analyzed with the capillary device. A linear relationship between the peak area and the concentration was demonstrated for the Fab at 3.2 pM to 10 nM. The coefficients of variation for the peak area and detection time at 10 nM were 4.3% and 4.4%, respectively. The coupled procedure described here allows total transfer of the specifically adsorbed proteins from the affinity column to the capillary for isoelectric focusing without any compromise in separation efficiency. Removal of excess salts and concentration of dilute samples are also attractive features of the coupled procedure that can provide a new option for the analysis of charge variants of a protein in biological samples.
固定化金属离子亲和色谱法与毛细管等电聚焦在单个毛细管中的直接耦合
描述了亲和色谱法和毛细管等电聚焦法在单个毛细管中的直接耦合过程。制作了统一的毛细管装置来实现这一过程。熔融二氧化硅毛细管的内表面在入口侧连续涂覆了螯合聚合物聚亚氨基二乙酸酯,在出口侧涂覆了中性聚合物聚二甲基丙烯酰胺。在用六组氨酸标签(模型样品)装载荧光标记的重组Fab后,用高盐缓冲液冲洗装置,然后用载体两性电解质溶液填充装置。用阳极溶液100mm磷酸填充镍螯合柱洗脱结合的Fab。在螯合柱一侧施加正电压,在同一侧施加压力,使酸化后的螯合柱产生的阳极电渗透压略过,使聚焦蛋白带逐渐通过固定式荧光检测器移动。将标记的重组Fab在变浓度下与标记的牛血清白蛋白在50 nM下混合,用毛细管仪进行分析。在3.2 pM至10 nM时,Fab的峰面积与浓度呈线性关系。10 nM峰面积和检测时间的变异系数分别为4.3%和4.4%。这里描述的耦合过程允许将特异性吸附的蛋白质从亲和柱完全转移到毛细管中进行等电聚焦,而不会影响分离效率。去除多余的盐和稀释样品的浓度也是耦合过程的吸引人的特点,可以为分析生物样品中蛋白质的电荷变异提供新的选择。
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