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Effects of trypsin to HEK293 cell membrane
Lipnická, Soňa ; Provazník, Ivo (referee) ; Čmiel, Vratislav (advisor)
This Bachelor’s thesis examines the effect of trypsin on the membranes of cell cultures. HEK293 cell lines nontransfected and cell lines stably transfected through the CaV3.1 membrane channels were selected for the optimization. They were gradually exposed to 0.25, 0.375 and 0.5 % trypsin. The concentration of trypsin influences the separation of the adherent cells on structure, as well as the degradation of cell membranes, which influences the cell cultures viability. Part of this thesis is an attempt to establish a methodology for the research of the effect of trypsin on membranes of HEK293 cell lines.
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Passive microrheology of colloidal systems based on biopolymers.
Bjalončíková, Petra ; Burgert, Ladislav (referee) ; Mravec, Filip (advisor)
Diploma thesis was aimed to deal with evaluation of microrheology method in the research of biopolymer-protein. Used biopolymer was sodium hyaluronate and proteins were trypsin and chymotrypsin. For measuring of microrheology were used particles with different radius (0,5 m and 1 m). It was found, that both substances have viscous charakter. Passive microrheology is suitable for measuring the viscoelastic properties of biopolymers.
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Characterization and stabilization of pancreatin
Wurstová, Agáta ; Němcová, Andrea (referee) ; Obruča, Stanislav (advisor)
This work focuses on a study of enzyme mixture pancreatin, its characterization and subsequent encapsulation into liposomes. As a reference proteins bovine serum albumin and trypsin were used. Characterization of pancreatin consisted of two parts. The first part focuses on optimization of methods for the concentration determination by absorption spectrophotometry using basic methods for identifying proteins (Biuret method, Hartree-Lowry method and Bradford method). Moreover, UV spectrums of the protein were measured. As a method for identification of protein´s molecular weight, SDS-PAGE was used. To identify components of pancreatin, LPLC was employed in two modifications, ion-exchange chromatography and size exclusion chromatography. The second part is dedicated to the characterization of pancreatin as enzyme in terms of pH and temperature optimum for the enzyme activities of protease (pH 9, 8 and 50 °C), amylase (pH 7 and 40 °C) and lipase (pH 7 and 50 °C). The last part of this work aimed at an encapsulation of pancreatin into liposomes and DLS analysis of distribution of particles and their zeta potential. Liposomes did not spontaneously release encapsulated enzyme. To confirm that proteins were successfully entrapped into liposomes, their structure was disrupted by application of phospholipase D. In conclusion, liposomes can be utilized as delivery systems for native enzymes.
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