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Capillary Sodium Dodecyl Sulfate Agarose Gel Electrophoresis of Proteins. | LitMetric

Capillary Sodium Dodecyl Sulfate Agarose Gel Electrophoresis of Proteins.

Gels

Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, 98 Nagyerdei Krt, H-4032 Debrecen, Hungary.

Published: January 2022

AI Article Synopsis

  • This study introduces capillary agarose gel electrophoresis as a new method for separating low molecular weight immunoglobulin subunits from monoclonal antibodies, which could improve existing techniques.
  • The research explores how varying the composition of agarose and boric acid in the gel affects the resolution and separation efficiency of these protein fragments, offering insights into the physical parameters at play.
  • The findings reveal that a specific gel composition (1% agarose/320 mM boric acid) achieves the best resolution for light and heavy chain fragments, while another composition (0.8% agarose/640 mM boric acid) offers superior separation power for fragments of similar molecular weight but different surface charges, without needing extensive maintenance between

Article 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, K) 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 (ΔK = 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 (ΔK = 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8871118PMC
http://dx.doi.org/10.3390/gels8020067DOI Listing

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