preparative Dotaz Zobrazit nápovědu
An improved preparative method based on isoelectric focusing of analytes in a cellulose-based separation medium is described in this study. Cellulose is suspended in an aqueous solution of simple buffers, ethylene glycol, glycerol, nonionic surfactant, and colored pI markers. Water partially evaporates during focusing run and the separation takes place in an in situ generated layer of cellulose, which has a gel-like appearance at the end of analysis. Final positions of analytes are indicated by the positions of zones of focused pI markers. Fractions, segments of the separation medium with analytes, can be simply collected by spatula and analyzed by downstream analytical methods. Good focusing ability of the new method and almost quantitative recovery of model proteins, cytochrome c and bovine serum albumin, was verified by gel electrophoresis and capillary isoelectric focusing of the collected fractions.
- Klíčová slova
- colored pI markers, isoelectric focusing, preparative, proteins, separation,
- MeSH
- celulosa * MeSH
- cytochromy c izolace a purifikace MeSH
- isoelektrická fokusace * MeSH
- pufry MeSH
- sérový albumin hovězí izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- celulosa * MeSH
- cytochromy c MeSH
- pufry MeSH
- sérový albumin hovězí MeSH
This study describes a new method for simultaneous identification of uropathogens in the case of polybacterial urinary tract infections. The method utilizes recently developed preparative isoelectric focusing (IEF) in cellulose-based separation medium with a subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative IEF was successfully used for both purification and separation of bacteria, Escherichia coli (pI 4.6) and Staphylococcus aureus (pI 3.4), in urine samples. The focused zones of bacteria, localized by the positions of focused colored pI markers, were easily collected from the separation media after the IEF analysis and then unambiguously identified by MALDI-TOF MS. The proposed method enables the identification of bacteria in urine specimens when the concentration of individual bacteria is ≥104 cells mL-1. Another benefit is the viability of bacteria extracted from the collected fractions after preparative IEF.
- Klíčová slova
- Bacteria, Infection, Isoelectric focusing, Mass spectrometry, Preparative, Urine,
- MeSH
- Escherichia coli izolace a purifikace MeSH
- infekce močového ústrojí mikrobiologie moč MeSH
- isoelektrická fokusace metody MeSH
- lidé MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Staphylococcus aureus izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Pre-separation and pre-concentration of bacteria is an important step especially when they are uncultured and bacterial concentration in the matrix is low. This study describes a preparative method based on isoelectric focusing of colored microorganisms in a cellulose-based separation medium from a high conductivity matrix. The isoelectric points found for the examined cells were 1.8 for Micrococcus luteus, 3.5 for Dietzia sp., and 4.7 for Rhodotorula mucilaginosa using capillary isoelectric focusing. The final positions of the zones of colored microbial cells in the cellulose-bed are indicated by colored pI markers. Segments of the separation medium with cells were harvested by a spatula, simply purified using centrifugation and analyzed by capillary isoelectric focusing and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The determined recovery ranged from 78% to 93%. The viability of the harvested cells was verified by their cultivation.
- Klíčová slova
- CIEF and MALDI-TOF MS, Colored microorganisms, Isoelectric points, Preparative isoelectric focusing,
- MeSH
- Actinobacteria izolace a purifikace MeSH
- celulosa chemie MeSH
- isoelektrická fokusace * MeSH
- Micrococcus izolace a purifikace MeSH
- Rhodotorula izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- celulosa MeSH
Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.
- Klíčová slova
- Insulin, Liquid chromatography-mass spectrometry, Preparative gel electrophoresis, Stem cell-derived islet organoids,
- MeSH
- chromatografie kapalinová MeSH
- elektroforéza v agarovém gelu MeSH
- inzulin * MeSH
- kapalinová chromatografie-hmotnostní spektrometrie * MeSH
- lidé MeSH
- organoidy MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inzulin * MeSH
During the last years we have witnessed progressive evolution of preparation of acenes with length up to dodecacene by on-surface synthesis in ultra-high vacuum or generation of acenes up to decacene in solid matrices at low temperatures. While these protocols with very specific conditions produce the acenes in amount of few molecules, the strategies leading to the acenes in large quantities dawdle behind. Only recently and after 70 years of synthetic attempts, heptacene has been prepared in bulk phase. However, the preparative scale synthesis of higher homologues still remains a formidable challenge. Here we report the preparation and characterisation of nonacene and show its excellent thermal and in-time stability.
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
With increasing demands on protein analyses in complex biological matrices, the insistence on developing new sample preparation techniques is rising. Recently, we introduced a new displacement electrophoresis technique (epitachophoresis) and instrumentation for preparative concentration and cleaning of DNA samples. This work describes the possibility of applying this device to protein samples. We have developed a method for the epitachophoretic concentration of proteins in a cationic mode and tested it by concentrating and collecting the protein zones from complex biological matrices (urine and growth medium). Under optimized conditions, we have obtained recoveries up to 99%. Furthermore, the applicability of the developed method was proven by concentrating and collecting the cytochrome c zone from a HeLa cell line growth medium, where the protein cytochrome c was released during cell apoptosis.
- Klíčová slova
- Concentration, Epitachophoresis, Growth medium, Proteins, Urine,
- MeSH
- cytochromy c MeSH
- HeLa buňky MeSH
- izotachoforéza * metody MeSH
- lidé MeSH
- proteiny MeSH
- tělesné tekutiny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cytochromy c MeSH
- proteiny MeSH
Collagens represent a large family (25 members identified so far) of closely related proteins. While the preparative procedures for the members that are ubiquitous and present in tissues in large quantities (typically fibre and network forming collagens types I, II, III, IV and V) are well established, the procedures for more recently discovered minor collagen types, namely those possessing large non-collagenous domain(s) in their molecule, are mostly micropreparative and for some collagenous proteins even do not exist. The reason is that the proof of their existence is based on immunochemical staining of tissue slices and nucleic database searching. Methods of preparation and identification of constituting alpha-polypeptide chains as well as collagenous and non-collagenous domains are also reviewed. Methods for revealing non-enzymatic posttranslational modifications (particularly of the fibre forming collagen types) are briefly described as well.
- MeSH
- chromatografie kapalinová metody MeSH
- kolagen chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- kolagen MeSH
We have developed a new separation device to concentrate and collect ions from several milliliter sample volumes to microliter fractions. Unlike most conventional platforms, this device has circular architecture. The electrophoretic migration operates from the outer perimeter toward the center. Separations can be performed both in continuous (zone electrophoresis) and discontinuous (moving boundary) electrolyte systems. We use a discontinuous electrolyte system comprising a leading and a terminating electrolyte to concentrate samples containing small organic anions and DNA fragment. The agarose gel stabilizes the boundary between the leading and terminating electrolytes. The milliliter volume sample is mixed with the terminating electrolyte and migrates through the gel toward the center. The concentrated total sample is collected in microliter fraction at the center. The potential for preparative concentration of DNA is demonstrated using a DNA ladder. Because zone migration accelerates as it moves toward the center, we named this method Epitachophoresis from the Greek word "επιταχυνω (epitachýnο)", meaning "acceleration". To the best of our knowledge, this unique circular architecture has not been previously described.
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Analytical and preparative high-performance liquid chromatography of 3 phenazines and furonaphthoquinone derivative on reversed-phase column are described. The mobile phase was methanol and water. The injected amount of the mixture was about 30 mg for a preparative chromatographic run requiring 80 min. Substances were detected directly in the column effluent by UV detection.
The preparative separation of an enzymatic hydrolysate by a step gradient of a lithium-sodium citrate buffer was studied. Using a two-column arrangement, 10 GBq of 14C-labelled enzymatic hydrolysate or 250 mg of inactive hydrolysate were separated within 36 h. After the chromatographic separation, the fractions of amino acids were isolated from the citrate eluents on weak cation exchangers with an ethylene glycol-poly(methyl methacrylate) matrix. The use of volatile organic acids as an eluent was possible due to the weak interactions with the cation matrix.
- MeSH
- aminokyseliny analýza MeSH
- hydrolýza MeSH
- proteinové hydrolyzáty analýza MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminokyseliny MeSH
- proteinové hydrolyzáty MeSH
