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Using of divergent flow isoelectric focusing and capillary liquid
Duša, Filip ; Moravcová, Dana ; Kahle, Vladislav ; Šlais, Karel
Separation of analytes from complex matrices play important role in their further identification. In this article we propose two-dimensional separation method of bovine serum albumin tryptic peptides. At the first dimension peptides are separated by divergent flow isoelectric focusing followed by reverse-phase liquid chromatography as the second dimension. Due to composition of input mixture it is possible to use autofocusing mode in first dimension. In this mode peptides are focused without addition of carrier ampholytes. Chromatograms from the second dimension were processed into contour map. Acquired data showed good separation efficiency of isoelectric focusing. Autofocusing mode enabled better resolution of peptides with close isoelectric points.
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Standards for capillary isoelectric focusing with laser induced fluorescence detection
Šlais, Karel ; Nováčková, J. ; Friedl, Z.
Proteins, peptides or BGE ampholytes labeled with suitable dyes were previously suggested as pI markers. However, both proteins and background ampholytes have many possible reactive sites for reactive dyes and this might result in formation of heterogeneously labeled product with respect to pI. By contrast, the low molecular pI markers can be prepared with heterogeneity and greater stability than proteins and could potentially be good pI markers as found in case of UV detection. To improve the laser induced fluorescence detection, new low molecular fluorescent compounds excitable around 490 nm with suitable acidobasic and electrophoretic properties were prepared and focused in capillary IEF with photometric or fluorometric detection. The experimental setup of IEF and properties of new laser induced fluorescent pI markers are given.
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Dynamic modification of proteins for fluorometric detection in CZE
Horká, Marie ; Šlais, Karel
The separation techniques employing fluorescence detection are sensitive and selective so they have been often applied for the trace analysis of biological samples. The commonly used derivatization of proteins can lower the detection limits; however, they can change the acido-basic properties and mobilities when compared to the native species. Recently, we have used the colored tenside as a buffer additive for the photometric detection of proteins in the UV region. The selectivity, efficiency and resolution of CZE separation using this dye were found to be similar to the CZE with SDS as the additive. In this study, the ampiphilic fluorescent compound is suggested as a buffer additive in CZE for dynamic modification of the sample of several proteins. Using the deuterium lamp for the excitation in the UV region for the on column fluorometric detection, the amol minimum detectable amounts of the proteins sampled on the CZE capillary were achieved.
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