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Use of fluorescence methods for the study of protein interactions
Johaníková, Klára ; Bezděková,, Jaroslava (referee) ; Pavelicová, Kristýna (advisor)
The diploma thesis "Use of fluorescence methods for the study of protein interactions" is focused on the use of fluorescence methods for the study of protein interactions using electromigration methods and Förster resonance energy transfer (FRET). The aim of this work was to create a bioconjugate of metallothionein (MT) protein with quantum dots (QDs) and commercial dyes. FRET was subsequently studied between these conjugates. QDs were synthesized under UV light and conjugation with MT was performed via a carbodiimide zerolengthcrooss-linker (EDC / sulfo-NHS), which serves to activate carboxyl groups and allows bioconjugation of the ligand by covalent bonding. Due to the high proportion of cysteines in MT, this protein has a very high affinity for metals. It is also involved in scavenging free radicals and there are studies that show that MT is overexpressed in cancer cells. Attention was also paid to the study of MT dimerization, which leads to an understanding of oxidative dimerization of MT and thus can contribute to understanding the formation of free radicals in the body and to deepen the knowledge about neurodegenerative disorders such as Parkinson's or Alzheimer's disease or amyotrophic lateral sclerosis. The formation of the MT dimer was confirmed by energy transfer between the donor (QDs) and the acceptor (commercial dye-cyanine) through the physical phenomenon of FRET and MALDI-TOF-MS.
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Study of metallothionein conjugates by electromigration methods
Johaníková, Klára ; Zemánková, Kristýna (referee) ; Vaculovičová, Markéta (advisor)
The bachelor thesis deals with the study of conjugates of metallothionein (MT) with fluorescent nanoparticles using electromigration methods. Particular attention is paid to the preparation and characterization of quantum dots (QDs) and their bioconjugates with the metallothionein protein, whose separation conditions (concentration and suitable electrolyte pH) have been carefully optimized. For the preparation of QDs, the action of UV radiation for one type of quantum dots and the thermal action of high temperature for the second type of species were used. Conjugation of QDs to the protein was performed via a carbodiimide-zero-length crosslinker (EDC / sulfo-NHS), which activates carboxyl groups and allows bioconjugation of the ligand by covalent bonding. The resulting bioconjugates were studied by capillary electrophoresis with absorption detection ( 214).
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Use of fluorescence methods for the study of protein interactions
Johaníková, Klára ; Bezděková,, Jaroslava (referee) ; Pavelicová, Kristýna (advisor)
The diploma thesis "Use of fluorescence methods for the study of protein interactions" is focused on the use of fluorescence methods for the study of protein interactions using electromigration methods and Förster resonance energy transfer (FRET). The aim of this work was to create a bioconjugate of metallothionein (MT) protein with quantum dots (QDs) and commercial dyes. FRET was subsequently studied between these conjugates. QDs were synthesized under UV light and conjugation with MT was performed via a carbodiimide zerolengthcrooss-linker (EDC / sulfo-NHS), which serves to activate carboxyl groups and allows bioconjugation of the ligand by covalent bonding. Due to the high proportion of cysteines in MT, this protein has a very high affinity for metals. It is also involved in scavenging free radicals and there are studies that show that MT is overexpressed in cancer cells. Attention was also paid to the study of MT dimerization, which leads to an understanding of oxidative dimerization of MT and thus can contribute to understanding the formation of free radicals in the body and to deepen the knowledge about neurodegenerative disorders such as Parkinson's or Alzheimer's disease or amyotrophic lateral sclerosis. The formation of the MT dimer was confirmed by energy transfer between the donor (QDs) and the acceptor (commercial dye-cyanine) through the physical phenomenon of FRET and MALDI-TOF-MS.
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Study of metallothionein conjugates by electromigration methods
Johaníková, Klára ; Zemánková, Kristýna (referee) ; Vaculovičová, Markéta (advisor)
The bachelor thesis deals with the study of conjugates of metallothionein (MT) with fluorescent nanoparticles using electromigration methods. Particular attention is paid to the preparation and characterization of quantum dots (QDs) and their bioconjugates with the metallothionein protein, whose separation conditions (concentration and suitable electrolyte pH) have been carefully optimized. For the preparation of QDs, the action of UV radiation for one type of quantum dots and the thermal action of high temperature for the second type of species were used. Conjugation of QDs to the protein was performed via a carbodiimide-zero-length crosslinker (EDC / sulfo-NHS), which activates carboxyl groups and allows bioconjugation of the ligand by covalent bonding. The resulting bioconjugates were studied by capillary electrophoresis with absorption detection ( 214).
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