Capillary Sodium Dodecyl Sulfate Agarose Gel Electrophoresis of Proteins

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.

Novel PCR-Based Multiplex Assays for Detecting Major Quality and Biotic Stress in Commercial and Weedy Rice

“…However, if funding is not a constraint, advanced genotyping methods such as capillary electrophoresis, real-time detection, or direct sequencing would be better alternatives, as they allow quick screening for alleles that differ by as little as 1 bp. The advanced methods are also slightly less labor-intensive because they can be automated and do not involve the use of agarose gel, although they require qualified laboratory personnel or skilled researchers to run the equipment and interpret the data [ 55 , 56 ]. Our study found that 89% of the tested weedy biotypes were resistant to bacterial leaf blight, indicating the potential to introduce resistance alleles from local weedy biotypes into cultivated rice varieties that are susceptible to the disease.…”

Analysis of Peptides and Proteins by Native and SDS Capillary Gel Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry via a Closed-Circuit Coaxial Sheath Flow Reactor Interface

“…Capillary electrophoresis (CE) is one of the frequently used liquid phase separation methods for the analysis of peptides and proteins, mostly utilizing UV or fluorescent detection both in zone and gel electrophoresis modes. , Hyphenation of CE with electrospray ionization mass spectrometry (ESI-MS), however, provides additional structural information about the separated sample components . In the past few decades, various CE-MS interfaces have been developed including sheath flow, sheathless, and liquid junction based approaches .…”

Capillary gel electrophoresis of very high molecular weight glycoproteins. Commercial and tailor-made gels for analysis of human monomeric and secretory immunoglobulin A

“…Capillary gel electrophoresis (CGE) with the use of polyacrylamide, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, dextran or agarose as sieving matrixes [1][2][3] is the CE mode of choice to analyze proteins according to their size. CGE has been thoroughly applied to the study of immunoglobulins (Ig) of the IgG class, mainly because of the importance of the IgG monoclonal antibodies (mAb) in the pharmaceutical field of the biologi-ily in their hinge regions (e.g., length of hinge region, disulfidebonding pattern, and the type and number of attached glycosylation sites).…”