Biomedical subjects

Milan J Benes

Publications and source records attributed to Milan J Benes.

7 recordsLinked to original sources

PubMed2005

Affinity chromatography of porcine pepsin A using quinolin-8-ol as ligand.

Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.

Animals↗

PubMed2005

Ferrite supports for isolation of DNA from complex samples and polymerase chain reaction amplification.

The influence of cobalt ferrite particles, with non-modified or modified surface, on the course of polymerase chain reaction (PCR) was investigated. DNA isolated from bacterial cells of Bifidobacterium bifidum was used in PCR evaluation of magnetic microspheres. The presence of cobalt ferrite particles inhibits PCR amplification. The effect is not dependent on the functional groups of the modifying reagents used (none, amino, carboxyl). Amplification was improved after the magnetic separation of magnetic particles. Proposed indirect method enabled verification of the suitability of designed particles for their application in PCR assays. Magnetic particles coated with alginic acid under high PEG and sodium chloride concentration were used for the isolation of PCR-ready bacterial DNA from various dairy products. DNA was isolated from crude bacterial cell lysates without phenol extraction of samples. Bifidobacterium and Lactobacillus DNAs were identified in dairy products using PCR.

Bifidobacterium↗

PubMed2005

Methacrylate-based chromatographic media.

This review summarizes the preparation and application of chromatographic separation media based on methacrylate monomers with a major focus on highly crosslinked macroporous beads prepared from 2-hydroxyethyl methacrylate and glycidyl methacrylate, respectively. The effects of process variables such as composition of the polymerization mixture that includes monomers, porogenic solvents, and free radical initiator, suspension stabilizer, reaction temperature, and stirring are detailed for both classical and templated suspension polymerization. In addition, specific features of the preparation of monodisperse beads are also discussed. The performance of methacrylate-based separation media is demonstrated on numerous separations in a variety of chromatographic modes.

Chromatography↗

PubMed2004

Isolation of genomic DNA using magnetic cobalt ferrite and silica particles.

Adsorption separation techniques as an alternative to laborious traditional methods (e.g., based on phenol extraction procedure) have been applied for DNA purification. In this work we used two types of particles: silica and cobalt ferrite (unmodified or modified with a reagent containing weakly basic aminoethyl groups, aminophenyl groups, or alginic acid). DNA from chicken erythrocytes and DNA isolated from bacteria Lactococcus lactis were used for testing of adsorption/desorption properties of particles. The cobalt ferrite particles modified with different reagents were used for isolation of PCR-ready bacterial DNA from different dairy products.

Adsorption↗

PubMed2004

Cleavage of double stranded plasmid DNA by lanthanide complexes.

The use of free lanthanide ions and their complexes for plasmid DNA pBR322 and chromosomal DNA cleavage was studied. Plasmid pBR322 DNA was treated by lanthanide chlorides (Eu(3+), La(3+), Nd(3+), Pr(3+), Gd(3+)) in HEPES buffer (pH 7.0, 7.5 and 8.0) at 24, 37, 50, 63, and 76 degrees C. The formation of linear and nicked plasmid forms was investigated depending on the reaction conditions. Heterogeneous lanthanide complexes of ethylenediamine tetraacetic acid (EDTA) immobilized on insoluble methacrylate support and iminodiacetic acid (IDA) immobilized on styrene support were used as catalysts plasmid for DNA pBR322 cleavage, too. The temperature of reaction mixture had substantial influence on cleavage rate. The precipitation of DNA occurred during the measurement of interactions between chromosomal DNA and La(3+) ions.

Chromosomes↗

PubMed2003

Chromatographic behaviour and purification of linear lambda phage and plasmid DNA molecules on 2-hydroxyethyl methacrylate-ethylene dimethacrylate-based supports.

The HEMA-BIO 1000 support, which is based on a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, was used for separation of lambda DNA and its fragments and plasmid pBR322 DNA. The separation of fragments greater than 6.6 kbp was demonstrated according to the slalom chromatography mechanism on column for size-exclusion chromatography in the case of linear lambda DNA fragments. The influence of particle size of column packing, mobile phase rate, and KCl concentration in mobile phase is discussed. The purification of plasmid DNA pBR322 using size-exclusion chromatography was more rapid compared to gel electrophoresis. The presence of salts in the eluate is not disadvantageous. DNA can be recovered from the eluate by ethanol precipitation. Plasmid DNA pBR322 isolated in this way was suitable for different biological applications (cleavage with restrictases, electrotransformation into bacterial cells).

Bacteriophage lambda↗

PubMed2002

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles.

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.

Cations, Divalent↗