Ramsay Lauren M, Dickerson Jane A, Dovichi Norman J
Department of Chemistry, University of Washington, Seattle, 98195-1700, USA.
Electrophoresis. 2009 Jan;30(2):297-302. doi: 10.1002/elps.200800498.
We have coupled CIEF with an LIF detector that is based on a post-column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the epsilon-amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503-labeled beta-lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.
我们已将CIEF与基于柱后鞘流比色皿的LIF检测器联用。在分析之前，我们使用Chromeo P503作为荧光试剂来标记蛋白质。该试剂与赖氨酸残基的ε-胺反应，保留残基的阳离子性质；标记后的蛋白质在CIEF中产生极其尖锐的峰。一组四种标准蛋白质在迁移时间和pI之间产生了线性关系。从巴雷特食管细胞系制备的蛋白质匀浆在40分钟的分离过程中分离出了100多种成分。注入毛细管的Chromeo P503标记的β-乳球蛋白的检测限为5 amol。两性电解质中存在的荧光杂质产生了很大的背景信号，与使用该检测器的其他形式的毛细管电泳相比，使检测限降低了四个数量级。
