Electrophoretic Separation and High-Sensitivity Detection of Dyestuff-Labeled Proteins Using an Untreated Fused-Silica Capillary and Sodium Dodecyl Sulfate-Containing Buffer for Migration and Labeling

Abstract

Proteins labeled with the dye Rhodamine B isothiocyanate were separated by capillary zone electrophoresis using an untreated fused-silica capillary and buffer containing sodium dodecyl sulfate. The sodium dodecyl sulfate in the migration buffer eliminated or minimized the protein adsorption to the inner wall of the capillary and rendered the proteins negatively charged through hydrophobic interactions. Furthermore, an appropriate use of SDS during the labeling procedure was found to increase the solubility of the dyestuff, which in turn results in sensitive detection of the protein. The labeled proteins were separated successfully in an untreated capillary using a buffer containing sodium dodecyl sulfate and detected spectrophotometrically and fluorometrically with high sensitivity. Bovine serum albumin reacted with the dyestuff for 4,12 and 24 h at 20° C was detected by spectrophotometry at 556 nm with approximately 2.5, 4.2 and 6.0 times, respectively, higher sensitivity than unlabeled protein. Fluorometric detection (λ_es=563 nm, λ_em= 577 nm) further increased the detection sensitivity to approximately twice that of spectrophotometric detection at 556 nm for the labeled protein.