|
|
Development of divergent-flow isoelectric focusing chip for separation of biological agents
Duša, Filip ; Šalplachta, Jiří ; Horká, Marie ; Lunerová, K. ; Rosenbergová, K. ; Kubíček, O.
Isoelectric focusing (IEF) is an efficient method for separation of different bacterium species. Divergent flow format of IEF (DF-IEF) enables continual separation of large volumes of samples and fractionation of the separated analytes for later analysis. The mentioned properties enabled development of DF-IEF chip for separation of H2O2 inactivated biological agents (B-agents). The chip was designed to perform separation in vertical alignment which solved issues connected with sedimentation of bacterial cells. The separation method was optimized using colored markers of isoelectric point and four different b-agents were separated using the DF-IEF chip. The obtained fractions were subsequently analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mass spectra proved that the developed DF-IEF chip is an effective tool for separation of inactivated B-agents.
|
|
| |
|
|
Study of transferrin as a marker of congenital disorders of glycosylation
Ondrušková, Nina ; Stiborová, Marie (advisor) ; Befekadu, Asfaw (referee)
Congenital disorders of glycosylation (CDG) represent a heterogeneous group of mul- tisystemic metabolic disorders which are caused by defects in biosynthetic pathways of glycoproteins. The screening test for N-glycosylation disorders is the analyses of sialylated isoforms of serum transferrin (Tf) by means of isoelectric focusing (IEF). Two distinct pathological IEF patterns of Tf are observed. A type I pattern is cha- racterized by a decrease of tetra- and an increase of di- and asialotransferrin, whereas a type II pattern shows in addition an increase of tri- and monosialotransferrin. The aims of diploma thesis were: 1) to evaluate reference range for spectrum of sialylated forms of Tf separated by IEF and 2) to perform biochemical and molecular analyses in three patients (P1-P3) with clinical suspicion for CDG. Serum and genomic DNA from three patients with clinical suspicion for CDG and family members of P1 were analysed. Sera from 99 healthy volunteers within the age range of 2-42 years served as a control group. Tf was analysed by IEF with direct immunofixation, SDS-PAGE and Western blot using specific antibody against human Tf (Dako). Profiles of Tf were quantified by AlphaEaseFC software (Alpha Innotech). Data were analysed by software STATISTICA 9.0 (StatSoft). TF a PMM2 genes were analysed...
|
|
|
The Determination and Comparison of the Electromigration Properties of Markers for Isoelectric Focusing
Lorinčíková, Kateřina ; Gaš, Bohuslav (advisor) ; Kašička, Václav (referee)
The dependencies of electrophoretic mobility on pH were measured for a set of 14 markers used for isoelectric focusing that were developed by the group of Šlais and that are based on substitutions on the nitrophenol core, and for a kit consisting of 5 pI markers developed by Shimura, which have an oligopeptide structure. The dissociation constants and limiting electrophoretic mobilities of these compounds were obtained from the dependencies with the use of the program AnglerFish. The isoelectric point values of the compounds were consequently calculated using the obtained data. A comparison of the obtained pI values with the values that have been declared in literature, albeit gained by different analytical methods, has been made. Key Words capillary zone electrophoresis, isoelectric focusing, pI markers, isoelectric point, thermodynamic dissociation constant, limiting ionic mobility
|
|
| |
|
| |
|
|
Study of transferrin as a marker of congenital disorders of glycosylation
Ondrušková, Nina ; Stiborová, Marie (advisor) ; Befekadu, Asfaw (referee)
Congenital disorders of glycosylation (CDG) represent a heterogeneous group of mul- tisystemic metabolic disorders which are caused by defects in biosynthetic pathways of glycoproteins. The screening test for N-glycosylation disorders is the analyses of sialylated isoforms of serum transferrin (Tf) by means of isoelectric focusing (IEF). Two distinct pathological IEF patterns of Tf are observed. A type I pattern is cha- racterized by a decrease of tetra- and an increase of di- and asialotransferrin, whereas a type II pattern shows in addition an increase of tri- and monosialotransferrin. The aims of diploma thesis were: 1) to evaluate reference range for spectrum of sialylated forms of Tf separated by IEF and 2) to perform biochemical and molecular analyses in three patients (P1-P3) with clinical suspicion for CDG. Serum and genomic DNA from three patients with clinical suspicion for CDG and family members of P1 were analysed. Sera from 99 healthy volunteers within the age range of 2-42 years served as a control group. Tf was analysed by IEF with direct immunofixation, SDS-PAGE and Western blot using specific antibody against human Tf (Dako). Profiles of Tf were quantified by AlphaEaseFC software (Alpha Innotech). Data were analysed by software STATISTICA 9.0 (StatSoft). TF a PMM2 genes were analysed...
|
|
| |
|
| |
|
|
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.
|
guest ::
