Automated ultra-thin-layer SDS gel electrophoresis of proteins using noncovalent fluorescent labeling

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Abstract

Ultra-thin-layer SDS gel electrophoresis in conjunction with automated laser-induced fluorescence detection is a novel and powerful method for the analysis of fluorophore-labeled proteins. The technique described in this paper employs instant, noncovalent fluorophore labeling by the addition of a fluorescent staining dye to the sample proteins either during or immediately prior to the sample loading process. Thus, the method does not require time- consuming post- or pre-separation staining/labeling. By combining the multilane format of SDS polyacrylamide slab gel electrophoresis and the high separation efficiency of capillary SDS gel electrophoresis, Ultra-thin-layer SDS gel electrophoresis features rapid, high-throughput, and high-resolution analysis of proteins in the molecular mass range of 14-116 kDa. The good heat dissipation inherent to the ultrathin format enables the use of agarose and agarose-based composite separation matrixes, which can be easily replaced within the separation platform. Labeling efficiency as a function of the concentration of the staining dye, SDS, and proteins is thoroughly discussed. Detection sensitivity of the method was found to be at the low-femtomole level (1.25 ng/band), determined by analyzing a set of serial dilutions of standard proteins. Practical example of molecular mass determination and characterization of a complex protein mixture are also shown.

Original languageEnglish
Pages (from-to)2519-2525
Number of pages7
JournalAnalytical Chemistry
Volume72
Issue number11
DOIs
Publication statusPublished - Jun 1 2000

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ASJC Scopus subject areas

  • Analytical Chemistry

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