Rapid analysis of covalently and non-covalently fluorophore-labeled proteins using ultra-thin-layer sodium dodecylsulfate gel electrophoresis

Gel electrophoresis is one of the most frequently used tools for the separa tion of complex biopolymer mixtures. In recent years, there has been consid erable activity in the separation and characterization of protein molecules by sodium dodecylsulfate (SDS) gel electrophoresis with particular interes t in using this technique to separate on the basis of size and to estimate molecular mass and protein purity. Although the method is informative, it i s cumbersome, time consuming and lacks automation. In this paper we report an automated, high-performance SDS gel electrophoresis system that is based on electric-field-mediated separation of SDS-protein complexes using an ul tra-thin-layer platform. The integrated fiber optic bundle-based scanning l aser-induced fluorescence detection technology readily provided high sensit ivity, real-time detection of the migrating solute molecules. Rapid separat ions of covalently and non-covalently labeled proteins were demonstrated in the molecular mass range 14 000 to 205 000 in less than 9 and 16 min, resp ectively. Excellent quantitation and lane-to-lane migration time reproducib ility were found for all the solute components using the multilane separati on platform. The limit of detection was found to be 1.5-3 ng/band for both labeling methods, with excellent linearity over a six times serial double-d ilution range. Molecular mass calibration plots were compared for both cova lently and non-covalently labeled proteins. A linear relationship was found between the molecular mass and electrophoretic mobility in the case of cov alently labeled samples, while a non-linear relationship was revealed for t he non-covalently labeled samples. (C) 2000 Elsevier Science B.V. All right s reserved.