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András Guttman - One of the best experts on this subject based on the ideXlab platform.
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Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis.
Current molecular medicine, 2021Co-Authors: K. Steven Cook, András Guttman, Jane Luo, Lawrence ThompsonAbstract:Background Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms, including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for the bulk release of plasmid products; thus, it should be analyzed accordingly. Methods and results The traditional analysis method for plasmid DNA is agarose Gel Electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduced a fast, sensitive, and robust plasmid analysis method using Capillary Gel Electrophoresis with laser-induced fluorescence detection (CGE-LIF). CGE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent DNA binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose Gel Electrophoresis. Conclusion As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CGE-LIF to monitor plasmid topology and possible degradation.
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The Effect of Temperature in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Protein Therapeutics.
Analytical chemistry, 2020Co-Authors: Csenge Filep, András GuttmanAbstract:The temperature-dependent migration of molecular weight protein size standards and several biotherapeutic proteins were studied in sodium dodecyl sulfate Capillary Gel Electrophoresis (SDS-CGE) in ...
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Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and Capillary Gel Electrophoresis ☆
Journal of chromatography. A, 2013Co-Authors: Márta Kerékgyártó, Zsolt Ronai, Nóra Németh, Tamás Kerekes, András GuttmanAbstract:The transmembrane protein wolframin (WSF1) plays a crucial role in cell integrity in pancreatic beta cells and maintaining ER homeostasis. Genetic variations in the WFS1 gene have been described to be associated with Wolfram syndrome or type 2 diabetes mellitus. In this paper we report on an efficient double-tube allele-specific amplification method in conjunction with ultrafast Capillary Gel Electrophoresis for direct haplotyping analysis of the SNPs in two important miRNA-binding sites (rs1046322 and rs9457) in the WFS1 gene. An automated single-channel Capillary Gel Electrophoresis system was utilized in the method that provided dsDNA fragment analysis in less than 240 s. The light-emitting diode induced fluorescence (LEDIF) detection system enabled excellent sensitivity for automated haplotyping of a large number of clinical samples. The detection limit was 0.002 ng/μL using field amplified injection from water diluted samples. The dynamic quantitation range was 0.08-10.00 ng/μL (R(2)=0.9997) in buffer diluted samples.
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Validation of a tentative microsatellite marker for the dopamine D4 receptor gene by Capillary Gel Electrophoresis
Journal of chromatography. A, 2006Co-Authors: Agnes Toth-petroczy, Zsolt Ronai, Ágnes Szilágyi, Maria Sasvari-szekely, András GuttmanAbstract:Two to four-basepair-short tandem repeats (i.e. microsatellites) are broadly utilized as genetic markers for mapping disease loci in whole genome search analyses. Based on their close vicinity on chromosome 11, the D11S1984 microsatellite was anticipated as a tentative marker for the dopamine D4 receptor gene. A Capillary Gel Electrophoresis based genotype analysis method and an in-house made computational tool was developed for the analysis of the D11S1984 microsatellite marker to examine a healthy Hungarian population of n=106. The data obtained did not suggest significant linkage between the D11S1984 marker and the DRD4 gene.
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Differential gene expression analysis by micro-preparative Capillary Gel Electrophoresis.
Journal of chromatography. A, 2003Co-Authors: András Guttman, Liang Shi, Julia Khandurina, Xun WangAbstract:Differential display analysis by cDNA fractionation, collection of differentially expressed fractions of interests and their downstream characterization is demonstrated. cDNA pools from two strains of Cochliobolus heterostrophus fungus were generated by specific restriction digestion and selective ligation. Micropreparative separation and isolation of differentially expressed transcript representatives were accomplished by high-performance Capillary Gel Electrophoresis. The collected individual DNA molecules were polymerase chain reaction amplified and sequenced to create expressed sequence tags for the genes of interests. High resolving power and sensitivity of Capillary Gel Electrophoresis enabled fast and automated processing of minute amounts of cDNA samples with high precision.
Edward Fritsche - One of the best experts on this subject based on the ideXlab platform.
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Determination of biotin on a protein by quantitative sodium dodecyl sulfate-Capillary Gel Electrophoresis of monomeric avidin.
Journal of Chromatography A, 2003Co-Authors: Huey G. Lee, Edward FritscheAbstract:Abstract Sodium dodecyl sulfate–Capillary Gel Electrophoresis (SDS–CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS–CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17 000. In the presence of a biotin–protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin and the initial amount is the portion of monomeric avidin bound to the conjugate. Accordingly, the number of biotin on the protein can be calculated. The assay is linearly responsive to increasing biotin loading in a biotinylation reaction of a protein. Accuracy of the assay is also demonstrated by good sample dilution recovery. Excellent quantitative reproducibility
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Rational approach to quantitative sodium dodecyl sulfate Capillary Gel Electrophoresis of monoclonal antibodies.
Journal of chromatography. A, 2002Co-Authors: Huey G. Lee, Steve Chang, Edward FritscheAbstract:Sodium dodecyl sulfate Capillary Gel Electrophoresis has been used to separate and quantify murine monoclonal antibodies. The method uses a murine IgG, whose subclass differs from the analyte antibody, as an internal reference. The internal reference is chosen based on knowing that mouse IgG1 can be separated from mouse IgG2a or IgG2b. Good intra- and inter-day reproducibility [relative standard deviation (RSD)
Nelson Cooke - One of the best experts on this subject based on the ideXlab platform.
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High-Resolution Capillary Gel Electrophoresis of Reducing Oligosaccharides Labeled with 1-Aminopyrene-3,6,8-trisulfonate
Analytical biochemistry, 1996Co-Authors: András Guttman, Fu Tai A. Chen, Ramon A. Evangelista, Nelson CookeAbstract:Abstract High-resolution Capillary Gel Electrophoresis was used for the separation of oligosaccharides labeled with a novel fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) at the reducing termini by reductive amination. The APTS–saccharide adducts were detected by laser-induced fluorescence with excitation by the 488-nm Ar-ion laser and a 520-nm emission filter. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to each molecule of oligosaccharide. Derivatization parameters, such as labeling reagent concentration, labeling temperature, and time as well as the influence of reaction solvent are thoroughly discussed. Desialylation of several sialylated oligosaccharides with different structures and linkages due to the effects of labeling temperature and time are also addressed. Employing the optimized conditions suggested in this paper, fluorophore labeling efficiency greater than 97% was achieved with no significant loss of sialic acid residues. The fluorescently labeled oligosaccharides are then separated and quantified by Capillary Gel Electrophoresis. Practical examples of low-level derivatization and high-resolution Capillary Gel Electrophoresis separation of N-linked glycans of ribonuclease-B and fetuin are also shown.
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Enhanced separation of DNA restriction fragments by Capillary Gel Electrophoresis using field strength gradients.
Analytical chemistry, 1992Co-Authors: András Guttman, Bart Wanders, Nelson CookeAbstract:The effect of electric field strength gradients on the separation of DNA restriction fragments was investigated. As reported in our earlier work, the mobility of different size double-stranded DNA molecules is a function of the applied electric field which suggests that the use of a nonuniform (time varying) electric field may increase the resolving power. We demonstrate that in Capillary Gel Electrophoresis enhanced separation of DNA restriction fragments up to 1353 bases pairs (bp) in size can be achieved by employing the field strength gradient method
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Prediction of migration behavior of oligonucleotides in Capillary Gel Electrophoresis.
Journal of chromatography, 1992Co-Authors: András Guttman, Robert J. Nelson, Nelson CookeAbstract:Abstract The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in Capillary polyacrylamide Gel Electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lenghts but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide Capillary Gel Electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10–20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.
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Effect of temperature on the separation of DNA restriction fragments in Capillary Gel Electrophoresis
Journal of Chromatography A, 1991Co-Authors: András Guttman, Nelson CookeAbstract:Abstract Two different separation modes were used in high-performance Capillary Gel Electrophoresis to study the effect of temperature on the separation of DNA restriction fragments, isoelectrostatic (use of constant applied electric field) and isorheic (use of constant current). In both instances, the migration properties and resolution of the DNA molecules were studied as a function of column temperature between 20 and 50°C. In the isoelectrostatic separation mode, the migration time and resolution decrease as temperature increases. In the isorheic separation mode, increasing the column temperature results in a maximum migration time for all of the DNA fragments and shows a maximum resolution for the lower molecular weight fragments (
Csenge Filep - One of the best experts on this subject based on the ideXlab platform.
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The Effect of Temperature in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Protein Therapeutics.
Analytical chemistry, 2020Co-Authors: Csenge Filep, András GuttmanAbstract:The temperature-dependent migration of molecular weight protein size standards and several biotherapeutic proteins were studied in sodium dodecyl sulfate Capillary Gel Electrophoresis (SDS-CGE) in ...
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Multilevel Capillary Gel Electrophoresis characterization of new antibody modalities
Analytica Chimica Acta, 1Co-Authors: Csenge Filep, Marton Szigeti, Robert Farsang, Markus Haberger, Dietmar Reusch, András GuttmanAbstract:Abstract Capillary Gel Electrophoresis-based methods were applied to comprehensively characterize two development phase new modality monoclonal antibodies including a glycoengineered and a bispecific test compound. The samples were subject to multilevel characterization at the intact (both by SDS-SGE and cIEF) as well as the reduced protein and the released N-glycan levels. SDS Capillary Gel Electrophoresis analysis showed excellent separation of the light and heavy chains of both samples. The bispecific antibody required a special temperature gradient denaturation process and a longer Capillary to resolve its two light chain fragments. Separation of PNGase F digested antibodies revealed migration time shifts, suggesting the presence of N-linked glycosylation on the corresponding subunits. For efficient glycan removal, the highly glycosylated glycoengineered monoclonal antibody was trypsin digested prior to the endoglycosidase treatment. The released glycans were profiled by Capillary Gel Electrophoresis after APTS labeling and their oligosaccharide structures were identified by exoglycosidase based carbohydrate sequencing. Finally, Capillary isoelectric focusing shed light on the charge heterogeneity of the test compounds, providing important complementary information. A flowchart was established for workflow optimization.
Huey G. Lee - One of the best experts on this subject based on the ideXlab platform.
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Determination of biotin on a protein by quantitative sodium dodecyl sulfate-Capillary Gel Electrophoresis of monomeric avidin.
Journal of Chromatography A, 2003Co-Authors: Huey G. Lee, Edward FritscheAbstract:Abstract Sodium dodecyl sulfate–Capillary Gel Electrophoresis (SDS–CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS–CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17 000. In the presence of a biotin–protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin and the initial amount is the portion of monomeric avidin bound to the conjugate. Accordingly, the number of biotin on the protein can be calculated. The assay is linearly responsive to increasing biotin loading in a biotinylation reaction of a protein. Accuracy of the assay is also demonstrated by good sample dilution recovery. Excellent quantitative reproducibility
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Rational approach to quantitative sodium dodecyl sulfate Capillary Gel Electrophoresis of monoclonal antibodies.
Journal of chromatography. A, 2002Co-Authors: Huey G. Lee, Steve Chang, Edward FritscheAbstract:Sodium dodecyl sulfate Capillary Gel Electrophoresis has been used to separate and quantify murine monoclonal antibodies. The method uses a murine IgG, whose subclass differs from the analyte antibody, as an internal reference. The internal reference is chosen based on knowing that mouse IgG1 can be separated from mouse IgG2a or IgG2b. Good intra- and inter-day reproducibility [relative standard deviation (RSD)
