Capillary Sodium Dodecyl Sulfate Agarose Gel Electrophoresis of Proteins

Daniel Sarkozy,Andras Guttman

doi:10.3390/gels8020067

Abstract

Capillary sodium dodecyl sulfate gel electrophoresis has long been used for the analysis of proteins, mostly either with entangled polymer networks or translationally cross-linked gels. In this paper capillary agarose gel electrophoresis is introduced for the separation of low molecular weight immunoglobulin subunits. The light (LC~24 kDa) and heavy (HC~50 kDa) chain fragments of a monoclonal antibody therapeutic drug were used to optimize the sieving matrix composition of the agarose/Tris-borate-EDTA (TBE) systems. The agarose and boric acid contents were systematically varied between 0.2–1.0% and 320–640 mM, respectively. The influence of several physical parameters such as viscosity and electroosmotic flow were also investigated, the latter to shed light on its effect on the electrokinetic injection bias. Three dimensional Ferguson plots were utilized to better understand the sieving performance of the various agarose/TBE ratio gels, especially relying on their slope (retardation coefficient, KR) value differences. The best resolution between the LC and non-glycosylated HC IgG subunits was obtained by utilizing the molecular sieving effect of the 1% agarose/320 mM boric acid composition (ΔKR = 0.035). On the other hand, the 0.8% agarose/640 mM boric acid gel showed the highest separation power between the similar molecular weight, but different surface charge density non-glycosylated HC and HC fragments (ΔKR = 0.005). It is important to note that the agarose-based gel-buffer systems did not require any capillary regeneration steps between runs other than simple replenishment of the sieving matrix, significantly speeding up analysis cycle time.

Highlights

Agarose is a linear polysaccharide consisting of repeating agarobiose units and a routinely used electrophoresis separation medium in bioanalytical and molecular biology laboratories
In this paper we evaluate the effect of agarose/borate concentration ratios on the electromigration properties of sodium dodecyl sulfate (SDS)–protein complexes, using the low molecular weight tromigration properties of SDS–protein complexes, using the low molecular weight light light and heavy chain subunits of a monoclonal antibody drug as model compounds
The electroosmotic flow (EOF) corrected effective electrophoretic mobility values were used to generate three dimensional Ferguson plots to better visualize the sieving behavior of the borate stabilized agarose gels

Summary

Introduction

Agarose is a linear polysaccharide consisting of repeating agarobiose units and a routinely used electrophoresis separation medium in bioanalytical and molecular biology laboratories. Agarose is one of the two major components of a mixture called agar and extracted from red algae by boiling, filtration, and freeze-thawing to remove impurities and the other main component of agaropectin [1,2]. As early as in 1949, Gordon et al used agar jelly for protein electrophoresis to separate ferritin from hemoglobin and to resolve egg white proteins [3]. Protein electrophoresis in unprocessed agar was most often compromised by adsorption of sample particles to, or precipitation in the gel [4]. These undesirable properties diminished when the agaropectin and other impurities were removed from the agar.

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