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A simple, rapid, and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate‐protein complexes by capillary electrophoresis with laser‐induced fluorescence
Author(s) -
Chiu TaiChia,
Lin YangWei,
Huang ChihChing,
Chrambach Andreas,
Chang HuanTsung
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305428
Subject(s) - capillary electrophoresis , chemistry , sodium dodecyl sulfate , chromatography , fluorescence , analyte , fluorescence spectroscopy , bovine serum albumin , laser induced fluorescence , analytical chemistry (journal) , electrolyte , sodium , capillary action , electrophoresis , materials science , physics , organic chemistry , electrode , quantum mechanics , composite material
Abstract 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% SDS and high concentrations of NaCl (100 m M ), these conditions are not appropriate to CE in view of Joule heating. To overcome that impediment, we applied a plug of 0.1% SDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 μL) prepared in TB buffer containing 50 m M 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 n M for bovine serum albumin and casein, respectively. With a linear dynamic range from 10 n M to 5 μ M , this method provides the capability of determining the concentration of casein in cow's milk as 0.45 ± 0.03 m M ( n = 5).