Isoelectric point marker Dotaz Zobrazit nápovědu
BACKGROUND: Successful implementation of capillary isoelectric focusing (cIEF) in general requires the use of reference points - markers of isoelectric point. The low-molecular-mass fluorescent compounds based on the fluorescein structure, capable of marking distinct isoelectric points (pIs) with good focusing ability, stability, high extinction coefficient, and fluorescence are an alternative to the peptide and protein pI markers. RESULTS: Four synthetic pathways were used to produce the ampholytic compounds and the structures along with their basic physical and chemical properties are given. Previously published low-molecular-mass pI markers were used to calibrate the candidate fluorescent compounds using capillary IEF (cIEF) with laser-induced fluorescence (LIF) detection. Linear and point-to-point pI calibration approach were compared for the pI evaluation. The results show 21 fluorescent pI markers (FPIMs). This group of markers covers pH range from 3.10 to 10.21. The usability of the developed FPIMs was further demonstrated by analysis of fluorescein isothiocyanate (FITC) labeled immunoglobin (IgG) standard. SIGNIFICANCE AND NOVELTY: pI markers are crucial for tracking the pH gradient and pI determination of biological ampholytic compounds. This paper presents and characterizes a novel pI marker set capable of tracing isoelectric points and supporting highly sensitive LIF detection-based applications. A basis of a newly developed cIEF-based fractionation method is described as well.
- Klíčová slova
- Capillary isoelectric focusing, Isoelectric point marker, Laser-induced fluorescence, Protein characterization,
- MeSH
- elektroforéza kapilární metody MeSH
- fluorescein * chemie MeSH
- fluorescence MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie MeSH
- isoelektrická fokusace * metody MeSH
- izoelektrický bod MeSH
- kapilární izoelektrická fokusace MeSH
- koncentrace vodíkových iontů MeSH
- lasery * MeSH
- molekulová hmotnost MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescein * MeSH
- fluorescenční barviva MeSH
Newly prepared azo compounds and several commercially available indicators were investigated for their applicability as colored isoelectric point (pI) markers for isoelectric focusing (IEF) in the acidic range below pH 5. The majority of compounds described here can serve as primary standards since their pI values were determined by UV-VIS spectrophotometry independently IEF and direct measurement with a pH electrode. Subjected to gel IEF they show narrow and well-observable zones of different colors. Finally, our work resulted in suggestion of a color ladder composed of pI markers covering the pH range from 1.5 to 4.7.
- MeSH
- azosloučeniny chemická syntéza chemie MeSH
- barvicí látky chemická syntéza chemie MeSH
- isoelektrická fokusace * MeSH
- izoelektrický bod MeSH
- koncentrace vodíkových iontů MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- azosloučeniny MeSH
- barvicí látky MeSH
We present a set of novel low-molecular-mass (LMM) compounds possessing ampholytic properties. The compounds were designed to perform as markers of isoelectric point (pI) in different isoelectric focusing (IEF) formats and feature direct detectability in UV and visible wavelength regions. Capillary isoelectric focusing (cIEF) was used to determine the purity of the focusing species and the compounds' pI values. Nitrophenol-based pI markers (NPIMs) published previously were used as standards for the pI value calibration. The presented compounds focused very well, but small portion of them contained focusing impurities, thus, we recommend them for use in other IEF formats like gel IEF and preparative IEF. Moreover, multi-wavelength detection enabled determination of individual markers based on their specific spectral profiles and different absorption at selected detection wavelengths in the electropherogram. The presented compounds compose a group of chemicals featuring excellent shelf stability and isoelectric focusing properties, inexpensive synthesis, universal/multimode detectability, and good solubility at pI. The presented results provide a solid ground for their use as reference standards in various isoelectric focusing methods.
- Klíčová slova
- Capillary, Colored, Focusing, Isoelectric point, Marker,
- MeSH
- isoelektrická fokusace metody MeSH
- izoelektrický bod MeSH
- koncentrace vodíkových iontů MeSH
- protonmotorická síla * MeSH
- pufry MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- pufry MeSH
Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n ≥ 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.
- Klíčová slova
- Capillary, Focusing, Isoelectric point, Marker, Protein,
- Publikační typ
- časopisecké články MeSH
Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.
Two-dimensional gel isoelectric focusing (2-D gel IEF) is presented as the combination of the same separation method used consecutively in two directions of the same gel. In this new method, after completion of IEF process in the first dimension the gel was cut into the separate strips, each containing selected analytes together with the appropriate part of the original broad pH gradient, and the strips were rotated by 90 degrees (with regard to the first IEF) and left to diffuse overnight. After diffusion the strips were subjected to the second IEF. During the second IEF, the corresponding narrow part of pH gradient in each strip was restored again, however, now along the strip. The progress of the separation process can be monitored visually by using colored low-molecular-weight isoelectric point (pI) markers loaded into the gel simultaneously with proteins. The unique properties of IEF, focusing and resolution power were enhanced by using the same technique twice. Two forms of beta-lactoglobulin (pI values 5.14 and 5.31, respectively) non-separated in the first IEF were successfully separated in the second dimension at relatively low voltage (330 V) with the resolution power comparable to the high-resolution gels requiring the high voltage during the run and long separation time. Glucose oxidase loaded as diluted solution into ten positions across the gel was finally focused into a single band during 2-D gel IEF. Since the first and second IEF are carried out on the same gel, no losses and contamination of analyte occur. The suggested method can be used for separation/fractionation of complex biological mixtures, similarly as other multidimensional separation techniques applied in proteomics, and can be followed by further processing, e.g., mass spectrometry analysis. The focusing properties of IEF could be useful especially in separation of mixtures, where components are at low concentration levels.
- MeSH
- 2D gelová elektroforéza metody MeSH
- isoelektrická fokusace metody MeSH
- koncentrace vodíkových iontů MeSH
- proteiny izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny MeSH
The research of microorganisms includes the development of methods for the inactivation of viruses and other microbes. It also means to efficiently eliminate the infectivity of microorganisms without damage of their integrity and structure. According to the results of the last 5 years the capillary electromigration techniques appear to be very perspective for the comparison of the methods applicable for inactivation in the diagnostics and study of the pathogens. In this paper we suggest the capillary isoelectric focusing of the model microorganisms, Escherichia coli, Staphylococcus epidermidis, Candida albicans and bacteriophage PhiX 174, native or inactivated by different procedures. UV detection and fluorometric detection for the dynamically modified microbes by pyrenebutanoate on the basis of the non-ionogenic tenside were used here. Isoelectric points of native and/or dynamically modified microorganisms and other properties were compared with those obtained after microorganisms inactivation. The segmental injection of the sample pulse enabled the reproducible and efficient capillary isoelectric focusing in different pH gradients. The low-molecular-weight pI markers were used for tracing of the pH gradient.
- MeSH
- Bacteria účinky léků MeSH
- bakteriofág phi X 174 účinky léků MeSH
- Candida albicans účinky léků MeSH
- elektroforéza kapilární metody MeSH
- Escherichia coli účinky léků MeSH
- isoelektrická fokusace metody MeSH
- izoelektrický bod MeSH
- mikrobiální viabilita MeSH
- pilotní projekty MeSH
- povrchově aktivní látky farmakologie MeSH
- Staphylococcus epidermidis účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- povrchově aktivní látky MeSH
This study describes a new method for fast identification of highly hydrophobic conidia of Aspergillus species from both simple and complex matrices. The method is based on recently developed preparative isoelectric focusing in a cellulose-based separation medium which had to be modified with respect to the highly hydrophobic surface of the conidia. Although Aspergillus conidia are colored, their zones in the cellulose bed were indicated by colored isoelectric point markers. The isoelectric point values of Aspergillus conidia were determined by capillary isoelectric focusing. Preparative isoelectric focusing was successfully used for preconcentration of individual conidia of cultivated strains of Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus parasiticus, and also for separation of the conidia in a mixture. Subsequently, red pepper powder and peanuts spiked with Aspergillus niger and Aspergillus flavus conidia, respectively, were used as complex matrices. The detection limit for identification of the conidia in these complex matrices is 104 conidia mL-1 . The presence of conidia in the focused zones was confirmed by their subsequent analysis by capillary isoelectric focusing. Their viability was confirmed by a cultivation of the conidia extracted from the collected fractions after preparative isoelectric focusing.
- Klíčová slova
- capillary isoelectric focusing, peanuts, red pepper powder,
- MeSH
- Arachis mikrobiologie MeSH
- Aspergillus chemie MeSH
- Capsicum mikrobiologie MeSH
- isoelektrická fokusace MeSH
- prášky, zásypy, pudry MeSH
- spory hub chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- prášky, zásypy, pudry MeSH
Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.
- MeSH
- isoelektrická fokusace metody normy MeSH
- koncentrace vodíkových iontů * MeSH
- pufry MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- pufry MeSH
Amphotericin B (AmB) is still, despite its severe nephrotoxicity, the first-line agent in the management of serious systemic fungal infections. A sensitive and reliable method is therefore required to control AmB concentration in body fluids of a patient. This study demonstrates the potential of the off-line combination of preparative isoelectric focusing (IEF) with capillary isoelectric focusing (CIEF) or capillary zone electrophoresis (CZE) in the determination of AmB in human blood serum. The required value of the isoelectric point of AmB was determined to be 6.1 using the CIEF technique. Preparative IEF served as a pre-separation and concentration technique. The pH gradient was traced by colored low molecular pI markers. The collected fraction with AmB was easily processed and then analyzed by CIEF and CZE. Tens of picograms of AmB in human blood serum sample can be determined by a combination of preparative IEF with CZE. The method was linear in the AmB concentration range of 0.3-600 ng mL-1. The recovery ranged from 93% to 98%.
- Klíčová slova
- Amphotericin B, Blood, Capillary zone electrophoresis, Isoelectric focusing, Sample preparation, Serum,
- MeSH
- amfotericin B krev izolace a purifikace MeSH
- biochemická analýza krve metody MeSH
- elektroforéza kapilární metody MeSH
- isoelektrická fokusace MeSH
- lidé MeSH
- limita detekce * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- amfotericin B MeSH
