Regulation of electroosmotic flow and electrophoretic mobility of proteinsfor concentration without desalting

Proteins were concentrated and separated in 0.6% poly(ethylene oxide) (PEO) solution using a capillary filled with Tris-borate (TB) buffer prior to an alysis and detected by laser-induced native fluorescence using a pulsed Nd: YAG laser. During the concentration and separation, PEO solution entered th e capillary by electroosmotic flow. When proteins dissolved in high salts ( phosphate-buffered saline) were separated using 0.6% PEO solution prepared in 200 mM TB buffer, pH 9.0, the limits of detection (LODs) at signal-to no ise ratios=3 for carbonic anhydrase (CA) and a-lactalbumin (a-lac) were on the levels of sub muM and muM, respectively. The LOD values compared to tho se obtained in 38 mM TB buffer were relatively high, which is likely due to salt quenching, Joule heating and poor stacking. To improve sensitivity fo r analysis of proteins in high-conductivity media, two on-line concentratio n approaches without desalting were developed. When using a capillary fille d with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 800 mM TB buf fer, pH 9.0, the LOD values for CA and alpha -lac were 13.8 nM and 126.0 nM , respectively, which were about 4.7 and 11.2-fold sensitivity enhancements compared to those obtained by a conventional hydrodynamic injection (30 cm height for 10 s), respectively. The sensitivity was further improved by in jecting a short plug of low pH buffer after protein injection using a capil lary filled with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 400 mM TB buffer, pH 9.0. A linear relationship between the peak height and th e injection volume up to 0.81 mul was obtained and the LOD values for CA an d ct-lac were down to 4.7 and 37.8 nM. (C) 2001 Elsevier Science B.V. All r ights reserved.