Utilization of micropreparative fast focusing by a new wide pH range electrolyte system based on bidirectional isotachophoresis

We suggest the possibility of practical utilization of a new electrolyte system for fast preparative focusing in wide pH range based on bidirectional isotachophoresis. The focusing occurs on nonwoven fabric strip positioned in an open horizontal V-shaped trough. It is based on bidirectional ITP with multiple counter ions and spacers created from commercially available simple buffers. Milk spiked with ampicillin was used as a sample and high performance liquid chromatography was as a second dimension for analysis of fast preparative focusing fractions. Speed, easy fraction handling, and possibility of pre-concentration of analytes from a raw sample are the benefits of this technique

Proteome analysis of Francisella tularensis bacterial mutant strains

Cílem diplomové práce byla komparativní proteomová analýza divokého kmene bakterie Francisella tularensis ssp. holarctica FSC 200 a mutantních kmenů s deletovanými geny ?pilC, ?pilQ, ?pilO1, ?pilO. Z uvedených bakteriálních kmenů byly připraveny jak celobuněčné lyzáty, tak i frakce obohacené o membránové proteiny. Všechny vzorky byly děleny 2D elektroforézou v rozmezí pH 3-10 a pH 6-11, digitalizovány pomocí softwaru KODAK 2D Image Analysis. Komparativní analýza mutantních kmenů a divokého kmene byla provedena pomocí programu ImageMaster 2D Platinum 6.0. Byly sledovány kvalitativní a kvantitativní změny proteinových skvrn a dále změny v posunu jejich isoelektrických bodů (?pI) a molekulových hmotností (?MW). Největší rozdíly v proteinové expresi byly nalezeny u mutantního kmene ?pilO2 a proto byla provedena jeho detailní analýza ukončená identifikací změněných proteinů. Identifikace proteinů byla provedena dvěma metodami: MALDI-TOF MS a komparativní analýzou s 2D referenčními proteinovými mapami. V rámci komparativní proteomové analýzy mutantního kmene ?pilO2 a mateřského kmene FSC200 byly pomocí MS identifikovány čtyři proteiny a pomocí srovnávací analýzy s referenční 2D mapou bylo identifikováno 21 proteinů. Vybrané proteiny budou zkoumány v dalších studiích.The aim of the thesis was a comparative proteome analysis of a wild strain bacteria Francisella tularensis ssp. holarctica FSC 200 and mutant strains with of deletion genes ?pilC, ?pilQ, ?pilO1, ?pilO2. Whole cell lysates and fractions enriched in membrane proteins were prepared from these strains. All samples were separated by 2D electrophoresis in range 3-10 and 6-11 pH gradients and digitized by KODAK 2D Image Analysis Softwar. Comparative analyses of the mutant strains and the wild strain were performed with the help of ImageMaster 2D Platinum 6.0 Software. Qualitative and quantitative changes of the protein spots and changes in shift of their isoelectric points (?pI) and molecular weights (?MW) were detected. The biggest differences in protein expression were found in mutant strain ?pilO2 and therefore its detailed analysis was performed and finished by identification of changed proteins. Identification of proteins was done by two methods: by MALDI-TOF MS and according to reference standard 2D maps. Four qualitative changed proteins from comparative proteome analysis of mutant strain ?pilO2 and wild strain FSC 200 were identified by MS and twendy-one quantitative changed proteins were identified according to reference standard 2D maps. Selected proteins will be examined in next studies.Katedra biologických a biochemických vědDokončená práce s úspěšnou obhajobo

Differences in fingerprints of biofilm-positive and biofilm-negative Candida strains exploitable for clinical practice

In this study, we have presented differences in fingerprints of Candida parapsilosis and Candida metapsilosis as well as their biofilm-positive and biofilm-negative forms. Fingerprints of yeasts were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis, capillary isoelectric focusing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Samples for analyses were prepared by treatment of yeasts by boiling water or ethanol. Fingerprints of tested yeasts cells have differed in all cases. This knowledge can be used for obtaining rapid and simple method which distinguishes above mentioned yeasts in standard biochemical laboratories

Differentiation of Candida strains by capillary HPLC

The aim of this study was to compare chromatographic profiles of lysate of Candida parapsilosis positive and Candida parapsilosis negative using capilary high performance liquid chromatography