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Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells
Ledvina, Vojtěch ; Klepárník, Karel
Caspases are proteases that play key role in the process of apoptosis, the programmed\ncell death. Among them, caspase-3 and -7 are main executioner caspases that cleave\nmany vital proteins during apoptosis and after their widespread activation, the process\ncannot be reversed. To analyze caspase-3/7 activation within single cells, a miniaturized\ndevice for parallel analysis of eight samples was developed. The assay is based on the\nmodified luciferin-firefly luciferase bioluminescence (BL) system. Individual\nsuspended cells were collected and transferred into detection microvials using a\nmicromanipulator. The bioluminescence was detected using a photon counting head\nwith cooled photcathode. The LOD suitable for detection of active caspase-3/7 in both\napoptotic and non-apoptotic cells was reached.
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Characterization of FRET sensor
Datinská, Vladimíra ; Klepárník, Karel ; Belšánová, B. ; Minárik, M. ; Foret, František
In this study, we present characterization of sensor based on Fӧrster resonance energy\ntransfer (FRET). The sensor is composed of ssDNA chain attached to a laboratory\nsynthesized quantum dot (QD). A complementary chain of a sample is labeled by a\nluminescent dye. When the dsDNA hybrid is formed, the energy from the QD (donor)\nis transferred to the dye (acceptor) and FRET is observed as a decrease of QD\nluminescence emission intensity and an increase of dye luminescence emission\nintensity.
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Development of Instrumentation and Methodology in Proteomic and Environmental Analysis
Hezinová, Věra ; Bobáľová, Janette (oponent) ; Lubal,, Přemysl (oponent) ; Čáslavský, Josef (oponent) ; Klepárník, Karel (vedoucí práce)
This work is focused on both targeted and comprehensive approaches of proteomic studies. The targeted proteomics brings information about protein presence and localization in a cell or tissue using luminescence labels based on quantum dots, while the comprehensive approach deals with the identification of changes in proteomes of two or more specimens of the same organism that have grown in various conditions. Since the proteomics requires high sensitive separation and identification techniques, various methods of sensitivity improvement in capillary electrophoresis - mass spectrometry were verified in this work. The use of a liquid junction interface as a hyphenation between these two techniques ensures a higher sensitivity of analyses. This was verified also by the analysis of ethanol and cocaine metabolites in human urine. The developed instrumentation and methodology are utilizable for the estimation of the influence of important environmental factors on living systems at the cellular as well as molecular levels.
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Quantum dot-based immunoprobe for optical and electrochemical detection
Dvořáková, V. ; Čadková, M. ; Datinská, Vladimíra ; Chałupniak, A. ; Korecká, L. ; Merkoci, A. ; Klepárník, Karel ; Foret, František ; Bílková, Z.
Three-stage labeling strategy allows the preparation of specific conjugates composed of required antibodies and quantum dots as an extremely sensitive bio-probe applicable in varied immunoassays with following optical and electrochemical detection. This approach is essentially based on carbodiimide chemistry however each single step of whole procedure is separated and controlled which makes this applied protocol oriented, highly efficient and it enables gain of pure antibody-conjugated quantum dots in ready-to-use condition.
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Simple route of caspase-3 FRET sensor synthesis using “click chemistry”
Lišková, Marcela ; Křenková, Jana ; 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 easily prepared Förster Resonance Energy Transfer (FRET). We use very simple chemistry called “click”. This type of chemistry takes advantages of quickness, simplicity and cheapness. “Click chemistry” is based on usage of various functional group and cross-linkers to combine individual molecules together.
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