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% SDS 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% SDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 microL) 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 microM, this method provides the capability of determining the concentration of casein in cow's milk as 0.45 +/- 0.03 mM (n = 5).
Chiu
Tai-Chia
TC
Department of Chemistry, National Taiwan University, Taipei, Taiwan, R.O.C.
Lin
Yang-Wei
YW
Huang
Chih-Ching
CC
Chrambach
Andreas
A
Chang
Huan-Tsung
HT
D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D005063:Europium; D005453:Fluorescence; D005470:Fluorometry; D007834:Lasers; D008892:Milk / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013997:Time Factors
D011506:Proteins; C436863:SYPRO Rose Plus; D012967:Sodium Dodecyl Sulfate; D005063:Europium