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Effect of the operational parameters on the electromigration of proteins in sodium dodecyl sulfate capillary gel electrophoresis in the presence of propidium iodide fluorescent dye.

Talanta

January 2025

Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary; Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, University of Debrecen, Hungary. Electronic address:

Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) is a frequently used analytical technique in size-based separation of proteins, playing a vital role in the biopharmaceutical industry for the analysis and characterization of therapeutic proteins, employing both UV and fluorescent detection. Understanding the effect of the operational parameters using easily applicable in migratio fluorescent labeling is increasingly critical, especially because multicapillary electrophoresis systems with fluorescent detection have recently gained prominence in high-throughput biopolymer analysis. In this study, the effects of the three most important user-adjustable operational parameters (temperature, gel concentration, and electric field strength) were investigated on the electrophoretic mobility and resolution of SDS-protein complexes in the presence of propidium iodide in the gel-buffer system.

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Computer simulation was utilized to characterize the electrophoretic processes occurring during the enantioselective capillary electrophoresis-mass spectrometry (CE-MS) analysis of ketamine, norketamine, and hydroxynorketamine in a system with partial filling of the capillary with 19 mM (equals 5%) of highly sulfated γ-cyclodextrin (HS-γ-CD) and analyte detection on the cathodic side. Provided that the sample is applied without or with a small amount of the chiral selector, analytes become quickly focused and separated in the thereby formed HS-γ-CD gradient at the cathodic end of the sample compartment. This gradient broadens with time, remains stationary, and gradually reduces its span from the lower side due to diffusion such that analytes with high affinity to the anionic selector become released onto the other side of the focusing gradient where anionic migration and defocusing occur concomitantly.

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Efficient capture of single-stranded DNA (ssDNA) is crucial for high-throughput sequencing, which influences the speed and accuracy of genetic analysis. Electrophoresis (EP) and electro-osmotic flow (EOF) have a significant impact on the translocation behavior of ssDNA through the nanopore. Experimentally, dynamically tracking these two effects remains challenging, and conventional numerical methods also struggle to capture their dynamic properties in the presence of DNA.

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