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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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Analysis of tissue-bound cells: Novel approaches using laser capture microdissection
Matalová, Eva ; Adamová, Eva ; Klepárník, Karel
Recent biomedical research prefers analysis of homogenous samples obtained from primary cells to cell lines. Moreover, attention is paid to cells of low number but high biological impact, such as signalling centres in embryonic development, stem cells or tumour cells. To localize cells of interest within an organ/tissue, histological sections are widely used. However, the methods of choice for further analysis of such samples are mostly limited to histochemistry, immunohistochemistry and in situ hybridization.
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New sensor for DNA mutation detection
Datinská, Vladimíra ; Klepárník, Karel ; Minárik, M. ; Foret, František
We present a design and synthesis of a new sensor intended for the genomic analysis in molecular cancer research. This sensor is based on the Förster resonance energy transfer (FRET) and can be used for the detection of complementary oligonucletide chains based on probe hybridization. Quantum dots (QDs) with their unique optical properties serve as suitable donors of energy in FRET.
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Multi-step synthesis of caspase-3 sensor based on Förster resonance energy transfer
Lišková, Marcela ; Klepárník, Karel ; Pazdera, P. ; Foret, František
Programmed cell death or apoptosis is regulated process of cell suicide. The central role in apoptosis play cysteine proteases called caspases. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues. Various techniques for the determination of caspase-3 are commercially available e.g. Enzyme Linked Immuno-Sorbent Assay (ELISA), Western blotting or flow cytometric analysis. The products of the cleavage can be detected by spectrophotometry, fluorimetry, chemiluminescence (CL) or ELISA. In this work, we suggested fluorescent sensor based on Förster Resonance Energy Transfer (FRET) to determine caspase-3 in cell nucleus or cytoplasm by apoptosis for very fast analysis by fluorescence microscopy without sample destroying.
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Optimization of N-glycopeptides analysis methods and their preliminary application to barley proteins study
Benkovská, Dagmar ; Flodrová, Dana ; Bobálová, Janette ; Laštovičková, Markéta
N-glycosylation is the most frequently studied plant protein post-translational modification. The analysis of Nglycopeptides after protein proteolytic digestion offers information about the structure of both oligosaccharide and peptide moiety. However, this method has so far been less commonly used. Since glycopeptides hardly ionize during MS analysis in the presence of non-glycosylated peptides, they need to be separated from the complex peptide mixture. In this study, the glycopeptides enrichment, purification and analysis methods were successfully optimized on two standard N-glycoproteins. Concanavalin A (ConA) lectin tips were used for glycopeptide capturing, and obtained fractions were purified on carbon tips and analyzed using MALDI-TOF mass spectrometry. The differences in the CID fragmentation of certain types of glycopeptides were found. This technique was then applied to glycopeptide analysis of barley grain and malt proteins. Several barley glycopeptides were found, however, their identification was very difficult. More proteins separation techniques will be required before this enrichment procedure in further studies.
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Integrated microfluidic device for droplet manipulation
Basova, E. ; Drs, J. ; Zemánek, J. ; Hurák, Z. ; Foret, František
Droplets based microfluidic systems have a big potential for the miniaturization of processes for bioanalysis. In the form of droplets, reagents are used in discrete volume, enabling high-throughput chemical reactions as well as single-cell encapsulation. Microreactors of this type can be manipulated and applied in bio-testing. In this work we present a platform for droplet generation and manipulation by using dielectrophoresis force. This platform is an integrated microfluidic device with a dielectrophoresis (DEP) chip. The microfluidic device generates microdroplets such as water in oil emulsion.
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Senzitive method of Caspase-3 detection in single stem cell
Adamová, Eva ; Klepárník, Karel ; Matalová, Eva
Caspases are involved in physiological process (e.g. cellular differentiation) but also can cause serious disorders. It is getting necessary to develop sensitive, miniaturized and fast methods amenable to analyze small amounts of samples. Luciferin/luciferase chemiluminescence reaction is one of the methods of caspase-3 detection. We have developed a miniaturized device enabling detection of caspase-3 and quantitation just in femtogram level (10–15 g) in single apoptotic human stem cells (neural crest derived). The technology is based on the specific cleavage of modified luciferin by caspase-3, emissions of photons and their detection by photomultiplier tube working in the photon counting regime.
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