A simple, rapid and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate-protein complexes by capillary electrophoresis with laser-induced fluorescence

We describe a segmental filling method for the analysis of SYPRO Red labeled sodium dodecyl sulfate (SDS)-proteins (SRSPs) by capillary electrophoresis-laser induced fluorescence (CE-LIF) with electroosmotic counterflow of poly(ethylene oxide) (PEO). It is shown that SDS and salt play a crucial role in determining the fluorescence intensity of the SRSP. Although the fluorimetric measurements reveal that the SRSPs fluoresce strongly in Tris-borate (TB) buffer containing 0.1% SIDS and high concentrations of NaCl (100 mm), these conditions are not appropriate to CE in view of Joule heating. To overcome that impediment, we applied a plug of 0.1% SIDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 muL) prepared in TB buffer containing 50 mm NaCl and SYPRO Red. When using a background electrolyte of 0.6% PEO in TB buffer containing NaCl, electroosmotic counterflow of the analytes allows one to concentrate large sample volumes (up to 1/3 of effective capillary length) in 21 min, with detection of 0.35 and 0.10 nm for bovine serum albumin and casein, respectively. With a linear dynamic range from 10 nm to 5 muM, this method provides the capability of determining the concentration of casein in cow's milk as 0.45 +/- 0.03 mm (n = 5).