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Vývoj čipu pro separaci biologických agens pomocí izoelektrické fokusace v rozbíhavém toku
Duša, Filip ; Šalplachta, Jiří ; Horká, Marie ; Lunerová, K. ; Rosenbergová, K. ; Kubíček, O.
Izoelektrická fokusace (IEF) je účinnou metodou schopnou separovat jednotlivé druhy bakterií. IEF v rozbíhavém toku (DF-IEF) umožnuje kontinuálně separovat větší objem vzorku a separované analyty jímat do frakcí k pozdější analýze. Těchto vlastností bylo využito při vývoji DF-IEF čipu pro separaci biologických agens (B-agens) inaktivovaných parami H2O2. Čip byl navržen pro separaci ve vertikální poloze, která zabránila sedimentaci běžně se vyskytující u bakteriálních vzorků a znemožňující účinnou separaci. Po optimalizaci separační metody za využití barevných markerů izoelektrického bodu byla provedena separace čtyř druhů B-agens. Všechny získané frakce byly dále analyzovány pomocí hmotnostní spektrometrie s matricí asistovanou laserovou desorpcí/ionizací (MALDI-MS). Hmotnostní spektra prokázala účinnost vyvinutého DF-IEF čipu pro separaci inaktivovaných b-agens.
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Evaluation of the 33 synthesized nitrophenol-based markers of isoelectic point
Duša, Filip ; Moravcová, Dana ; Šlais, Karel
33 nitrophenol-based compounds, which absorb at ultraviolet as well as visible wavelengths, were selected as candidates for marking isoelectric points (pI) over the pH range from 3 to 10. The evaluation and selection of the candidate structures included structure-based calculations of acid-base properties. The selected compounds were further tested experimentally using capillary isoelectric focusing. The experimental analysis excluded two compounds due to producing double peaks during the analysis. Overall, 31 novel compounds were identified as suitable pI markers.
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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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