Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography
Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography
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DOI：
10.1007/s00216-011-5670-3
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发表时间：
2012-01
影响因子：
4.3
通讯作者：
J. Sproß;A. Sinz
中科院分区：
文献类型：
--
作者：
J. Sproß;A. Sinz
在100 μm内径的熔融石英毛细管中制备了聚（甲基丙烯酸缩水甘油酯-co-丙烯酰胺-co-二甲基丙烯酸乙烯酯）和聚（甲基丙烯酸缩水甘油酯-co-二甲基丙烯酸乙烯酯）整体柱，并采用戊二醛技术用单体亲和素修饰。用生物素（5-荧光素）偶联物以及生物素和荧光素标记的牛血清白蛋白（BSA），通过荧光光谱法测定了固定化单体亲和素（MACMA）的整体亲和柱的生物素结合能力。亲和柱能够分别结合16.4和3.7 μmol生物素/mL。使用聚（甲基丙烯酸缩水甘油酯-共-二甲基丙烯酸乙烯酯）整料制备的柱保留7.1 mg BSA/mL，几乎是市售单体抗生物素蛋白珠的6倍。基于MALDI-TOF质谱监测的方案被优化用于生物素化蛋白质和肽的富集。MACMA和市售单体抗生物素蛋白珠之间的富集效率的比较产生了我们的新型整体亲和柱的上级结果。然而，在这项工作中提出的亲和介质遭受了显着程度的非特异性结合，这可能会妨碍更复杂的混合物的分析。进一步修改的整料的表面，设想未来的发展整料与改进的富集特性。
A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.