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IEF and HPLC-based methods for efficient bacterial characterization
Kubesová, Anna ; Šalplachta, Jiří ; Moravcová, Dana ; Horká, Marie
Pectobacterium and Dickeya species are responsible for blackleg and tuber soft rot diseases on crop and ornamental plants. Recently, a more virulent and aggressive Dickeya type of E. chrysanthemi was described and it becomes serious problem for potato production in Europe. In this study, we suggest procedures using two commonly available laboratory techniques, gel isoelectric focusing and liquid chromatography, for differentiation and characterization of Pectobacterium and Dickeya species. We have confirmed that fingerprinting approach can be used for bacterial differentiation, which can be further used in rapid diagnosis of plant disease.
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New isoelectric focusing power supply based on features of voltage multiplier
Duša, Filip ; Šlais, Karel
Present electrophoretic separation methods rely on sophisticated high-voltage power supplies capable of programing a voltage / current time course during an analysis. In this paper we suggest design of a simple highvoltage power supply for isoelectric focusing composed from affordable and commonly available electrical parts. It is based on features of the voltage multiplier invented by Cockcroft and Walton. Electrical characteristics of the power supply enabled power load controlled isoelectric focusing analysis thus eliminating need for programing voltage time course and reducing analysis total time.
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Micropreparative solution isoelectric focusing of peptides and proteins in nonwoven strip
Duša, Filip ; Šlais, Karel
Recently we devised a new instrument for micropreparative analysis [1]. It is based on solution phase isoelectric focusing (sIEF) performed in a narrow channel (approximately 2 – 4 mm wide) with a strip of nonwoven fabric serving as the analysis bed. The strip can be precut before an analysis to make harvesting of fractions easier. Isoelectric focusing is driven by a programmable electrophoretic power supply. Progress of analysis is monitored by addition of colored isoelectric point (pI) markers. Usually sIEF runs are performed overnight. Evaporation of water is one of crucial features of analysis and it forces free liquid to shrink into the nonwoven fabric strip. Moreover, water evaporation increases liquid viscosity by altering ethylene glycol/water ratio. Fractions can be harvested simply by collecting and washing precut segments after the sIEF run. In this paper we used sIEF device for purification of caseinomacropeptide (CMP) from crude whey. Obtained fractions were further analyzed by HPLC. From acquired data a plot with chromatograms of all fractions was constructed to show purification profile of CMP. We proved that the new instrument is capable of quantitative purification of CMP complex from a globulin fraction.
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