Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Anal Bioanal Chem. 2011 Jun;400(7):2025-30. doi: 10.1007/s00216-011-4926-2. Epub 2011 Apr 2.
Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates. This poor reproducibility appears to be associated with erratic variations in electroosmotic flow. One cause of electroosmotic flow variation is degradation of the capillary coating caused by the extremely basic solution commonly used during mobilization and focusing; this degradation of the capillary coating can be reduced by employing a CAPS mobilization buffer at pH 9. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. We report the use of CAPS mobilization buffer in combination with an ultrasensitive laser-induced fluorescence detector for the reproducible analysis of ∼2 ng of protein from a Barrett's esophagus biopsy.
毛细管等电聚焦往往存在重现性差的问题，特别是在分析细胞或组织匀浆中的复杂蛋白质样品时。这种重现性差似乎与电渗流的不稳定变化有关。电渗流变化的一个原因是由于在迁移和聚焦过程中常用的极其碱性溶液导致毛细管涂层的降解；通过使用 CAPS 迁移缓冲液（pH9）可以减少毛细管涂层的这种降解。另一个变化的原因是蛋白质吸附到毛细管壁上，这会导致电渗流增加。通过在缓冲液中使用表面活性剂和采用极低的样品加载量，可以减少蛋白质吸附的影响。我们报告了使用 CAPS 迁移缓冲液与超灵敏激光诱导荧光检测器相结合，可从 Barrett 食管活检中重现性地分析约 2ng 的蛋白质。
