On-Line Concentration of a Protein Using Denaturation by Sodium Dodecyl Sulfate
On-Line Concentration of a Protein Using Denaturation by Sodium Dodecyl Sulfate
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DOI：
10.2116/analsci.21.37
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发表时间：
2005-01
影响因子：
1.6
通讯作者：
P. Jing;T. Kaneta;T. Imasaka
中科院分区：
文献类型：
--
作者：
P. Jing;T. Kaneta;T. Imasaka
描述了一种新的蛋白质柱上样品堆叠方法。该策略利用了蛋白质分子和十二烷基硫酸钠（SDS）单体之间的相互作用。将蛋白质样品（酸性或碱性）的长塞注入充满含有SDS的背景电解质（BGE）的毛细管中。当施加电位时，蛋白质与BGE中的SDS单体相互作用，形成蛋白质-SDS复合物，其迁移速度比相应的未络合蛋白质慢，导致蛋白质堆积。酸性蛋白质和碱性蛋白质在堆叠后的电泳速度几乎相同，这表明在BGE区形成的蛋白质- sds复合物具有相似的电荷/质量比。用一种碱性蛋白、溶菌酶和一种小分子亚甲基蓝组成的样品研究了堆积机理。研究结果清楚地表明，与SDS发生了两种相互作用，一种是蛋白质分子与SDS单体之间的逐步结合相互作用，另一种是小分子进入SDS分子形成的胶束的相互作用。该方法也适用于用荧光标记试剂在痕量水平标记的蛋白质的检测。即使在标记效率过低而无法通过短塞注射检测的标记条件下，也能检测到所标记的蛋白。
A novel method for the on-column sample stacking of proteins is described. The strategy takes advantage of interactions between protein molecules and sodium dodecyl sulfate (SDS) monomers. A long plug of a protein sample (either acidic or basic) is injected into a capillary filled with a background electrolyte (BGE) containing SDS. When a potential is applied, the proteins interact with SDS monomers in the BGE to form protein-SDS complexes that migrate more slowly than the corresponding uncomplexed protein, resulting in protein stacking. Both acidic and basic proteins migrate at an almost identical electrophoretic velocity after stacking, which indicates that the protein-SDS complexes formed in the BGE zone have a similar charge/mass ratio. The mechanism of stacking was investigated using a sample consisting of a basic protein, lysozyme, and a small molecule, methylene blue. The findings clearly show that two interactions with SDS occur, a stepwise binding interaction between protein molecules and SDS monomers and an interaction in which the small molecules enter into micelles formed by SDS molecules. The method was also applied to the detection of a protein labeled with a fluorescent labeling reagent at trace levels. The labeled protein was detected even under labeling conditions where the labeling efficiency was too low to detect by short-plug injection.