On-line concentration of proteins by SDS-CGE with LIF detection.
We present a simple approach for on-line concentration of SDS-protein complexes by using poly(vinyl alcohol) (PVA) solution in CGE. In comparison to the coated capillary, the presence of EOF in CGE omitted the need to fill the capillaries with polymer solutions prior to the analysis. More importantly, we found that highly reproducible separation of eight proteins by 3.5% PVA was achieved between runs and without the regeneration of high bulk EOF; the RSD of migration times was less than 0.7%. To further improve the concentration sensitivity, neutral PVA was introduced into the capillary with the help of EOF to act as sieving matrix. The occurrence of stacking at the boundary between the PVA and the sample zone is mainly due to the retardation of proteins by PVA. As a result, the LODs at an S/N of 3 for SDS-protein complexes are of the order of sub-nM to several nM. For example, the LOD for BSA is 0.78 nM, which is a 91-fold sensitivity enhancement over the normal injection. In addition, our stacking method has been applied to the analyses of proteins in Escherichia coli cells. The peak for beta-galactosidase (E. coli) was observed after 0.1 microM beta-galactosidase was spiked into the E. coli samples.
Yu
Cheng-Ju
CJ
Department of Chemistry, National Sun Yat-sen University, Taiwan.
Chang
Hui-Chiu
HC
Tseng
Wei-Lung
WL
D053841:Electroosmosis; D019075:Electrophoresis, Capillary / Q000379:methods*; D029968:Escherichia coli Proteins / Q000302:isolation & purification; D005453:Fluorescence; D007834:Lasers; D009862:Online Systems; D011142:Polyvinyl Alcohol; D011506:Proteins / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; D011506:Proteins; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D011142:Polyvinyl Alcohol