This work describes the SDS-CGE of proteins carried out in microchannels made of the negative photoresist EPON SU-8. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology allows the monolithic integration of the electrodes in the device. A high wafer fabrication yield and mass production compatibility guarantees low costs and high reliability. A poly(methyl methacrylate) (PMMA) packaging allows an easy setup and replacement of the device for electrophoresis experiments. In addition, the wire-bonding step is avoided. The electrophoretic mobilities of four proteins have been measured in microchannels filled with polyacrylamide. Different pore sizes have been tested obtaining their Ferguson plots. Finally, a separation of two proteins (20 and 36 kDa) has been carried out confirming that this novel device is suitable for protein separation. A resolution of 2.75 is obtained. This is the first time that this SU-8 microfluidic technology has been validated for SDS-CGE of proteins. This technology offers better separation performance than glass channels, at lower costs and with an easy packaging procedure.
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http://dx.doi.org/10.1002/elps.200600103 | DOI Listing |
Anal Chem
July 2024
Translational Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem 8200, Hungary.
Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis.
View Article and Find Full Text PDFSci Rep
November 2023
Department of Chemistry, State University of New York, Potsdam, NY, 13676, USA.
Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or isoferritins) with different proportions of H and L subunits that contribute to their structural and compositional heterogeneity, and thus physiological functions. Using size exclusion and anion exchange chromatography, capillary isoelectric focusing (cIEF), and SDS-capillary gel electrophoresis (SDS-CGE), we reveal for the first time a significant variation in ferritin subunit composition and isoelectric points, in both recombinant and native ferritins extracted from animal organs.
View Article and Find Full Text PDFAnal Chem
September 2022
Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, H-4032 Debrecen, Hungary.
The electromigration dispersion of the light- and heavy-chain subunit peaks of the therapeutic monoclonal antibody omalizumab was investigated in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran sieving matrices. Increasing boric acid content (340-640 mM) caused electromigration dispersion shifts for both low (2%)- and high (10%)-dextran-concentration gels in all gel-buffer compositions. In case of the heavy-chain fragment, elevated borate concentrations resulted in decreasing tailing and increasing fronting with the use of higher- and lower-dextran-concentration gels, respectively.
View Article and Find Full Text PDFFront Bioeng Biotechnol
March 2022
Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Molecular Medicine, University of Debrecen, Debrecen, Hungary.
Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5-10% hospitalization and 2-3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell the angiotensinconverting enzyme 2 receptor.
View Article and Find Full Text PDFJ Pharm Biomed Anal
May 2022
SCIEX, 250 S. Kraemer Blvd, Brea, CA 92821, USA.
A 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection (NFD) of proteins in capillary electrophoresis. The NFD scheme was evaluated in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) for monoclonal antibody (mAb) characterization. Utilizing a technique by which we filtered the LED emission through a 280 nm bandpass filter, we were able to increase overall concentration sensitivity of SDS-CGE-NFD ~2.
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