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Časopis/zdroj: Journal of biochemical and biophysical methods

21 záznamů v Medvik Filtry

Článek

Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins

Journal of biochemical and biophysical methods. 2008 ; 70 (6) : 1091-1097.

J Biochem Biophys Methods
ISSN 0165-022X
Medvik
Zdroj

The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.

Článek

Hydroxymethyl methacrylate-based monolithic columns designed for separation of oligonucleotides in hydrophilic-interaction capillary liquid chromatography

Journal of biochemical and biophysical methods. 2007 ; 70 (1) : 23-29.

ISSN 0165-022X
Medvik
Zdroj

Hydroxymethyl methacrylate-based monolithic columns for separation of oligonucleotides by capillary liquid chromatography (CLC) were prepared. We optimized composition of the polymerization mixture, which contained the monomer mixture consisting of N-(hydroxymethyl) methacrylamide (HMMAA) and ethylene dimethacrylate (EDMA), and the porogenic system composed of propane-1-ol, butane-1,4-diol and alpha, alpha'-azoisobutyronitrile (AIBN) as initiator. Separations of oligonucleotides were performed in HILIC (hydrophilic-interaction) mode using 100 mM triethylamine acetate (TEAA) in acetonitrile and in water as eluents. The influence of steepness of the mobile phase gradient on separation of the oligonucleotides was evaluated as well as the reproducibility of HMMAA monolith preparation.

Článek

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Journal of biochemical and biophysical methods. 2007 ; 70 (3) : 511-513.

ISSN 0165-022X
Medvik
Zdroj

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.

Článek

Separation of bacteria in temperature gradient: micro-thermal focusing field-flow fractionation

Journal of biochemical and biophysical methods. 2007 ; 70 (4) : 685-687.

ISSN 0165-022X
Medvik
Zdroj

The separation of Staphylococcus epidermidis and Rhodococcus erythropolis bacteria was achieved with the use of Micro-Thermal Focusing Field-Flow Fractionation. This is the first performance of separation exploiting the Ludwig-Soret effect (thermal diffusion) of living biological cells, combined with lift forces and resulting in the focusing mechanism of separation. The experiments were carried out under carefully chosen experimental conditions preventing the denaturation of the bacteria.

Článek

Effect of the matrix structure and concentration of polymer-immobilized Ni2+-iminodiacetic acid complexes on retention of IgG1

Journal of biochemical and biophysical methods. 2006 ; 67 (1) : 7-25.

ISSN 0165-022X
Medvik
Zdroj

Terpolymer bead particles (100-350 microm in diameter) were prepared by suspension radical polymerization from methacrylate esters [2,3-epoxypropyl methacrylate (GMA), 2-(2-hydroxyethoxy)ethyl methacrylate (DEGMA) and ethylene dimethacrylate (EDMA)] and subsequently derivatized affording iminodiacetic acid (IDA) chelating sorbents. The sorbents differed in pore volumes (0-0.7 cm3/g) and specific surface areas (0.03-9.8 m2/g) of their matrices as well as in the amounts of immobilized Ni2+-IDA complexes (0.03-1.58 mmol/g). The binding of imidazole was studied by frontal chromatography to evaluate the accessibility of Ni2+-IDA complexes. It was found that an increase in the bonded imidazole content with increasing immobilized Ni2+-IDA concentration was strongly dependent on the matrix morphology. A higher pore volume of the matrix significantly improved the utilizability of Ni2+-IDA complexes for imidazole binding. The performance of the sorbents based on two porous matrices with immobilized Ni2+-IDA concentration (0.1-1.58 mmol/g) differing in pore size distributions was compared in immobilized metal affinity chromatography (IMAC) of monoclonal mouse immunoglobulin IgG1 specific against human choriogonadotropic hormone (GTH-spec IgG1). The results have shown that sorbents based on matrix with large pores (up to 20 microm in diameter) exhibited high protein binding capacities. The GTH-spec IgG1 (Mw=158,000) was eluted from all the sorbents in its native form as was confirmed by MALDI-TOF.