Staining Method for Protein Analysis by Capillary Gel Electrophoresis

Abstract

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS-protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS-proteindye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS-protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/ mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most commonly used methods for protein analysis. Usually, a staining and destaining process is involved in order to properly detect the separated protein bands. Capillary electrophoresis (CE), and more recently microchip-based electrophoresis, is a miniaturized electrophoresis technique that has many advantages (e.g., high separation efficiency, short separation time, low mass detection limit, on-line detection, and automated operation) over traditional slab gel techniques. SDS-PAGE has been performed in capillaries 1-5 and on microchips. 6-9 Unfortunately, the staining and destaining process used in conventional slab gels cannot be simply adopted in CE. Typically, the separated proteins are detected in-column by either an ultraviolet (UV) absorbance or a laser-induced fluorescence (LIF) detection system. UV absorption is arguably the most frequently used detection mode in CE. It is also commonly employed in capillary SDS-PAGE, 1,2 since protein-SDS complexes absorbs light around 280 nm due to the aromatic side groups of amino acids and around 200−220 nm due to the peptide bonds between amino acids. LIF detection is preferred when low limit of detection (LOD) and wide linear dynamic range are desired. Native fluorescence of proteins has been explored for direct protein detection, 10 but it is not widely accepted because of the use of expe...

Capillary Electrophoresis in Bioanalysis

“…A fluorescent surfactant (F-16) that has a fluorescein head-group bound to a hexadodecyl alkyl chain was used to enhance the sensitivity of a LIF detection scheme of proteins that are separated in the presence of SDS (119). Proteins labeled with F-12 were size-based separated as cocomplexes of SDS-protein-F-12 in polyacrylamide-filled capillaries; limits of detection were 0.13 ng/mL BSA, dynamic range is 5 orders of magnitude, and fluorescence response is proportional to the square root of the protein concentration.…”

Protein separation by capillary gel electrophoresis: A review

“…Chiu et al [74] labeled proteins with SYPRO Red and accomplished LIF detection using a low-cost He-Ne laser. In 2007, Wu et al [113] developed an elegant approach for protein labeling. First a pseudo SDS dye was synthesized by attaching an alkyl chain to an ionic fluorescent dye (e.g., FITC).…”

Dilution of protein‐surfactant complexes: A fluorescence study

“…Although, the dynamic and kinetics of surfactant‐protein interactions has been studied extensively, to the best of our knowledge, the effect of dilution on the fluorescence intensity of protein–surfactant complexes has not been studied. Moreover, a great volume of literature on interaction of protein and surfactants is focused on single protein Bovine Serum Albumin (BSA) . Due to the wide range of differences in protein structure, there is a need for analyzing these interactions in a system of multiple proteins with different molecular weights.…”