2D electrophoresis
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Several new fast staining protocols for the visualization of proteins separated by SDS-PAGE utilizing Coomassie Blue staining (CBS) have been described in literature. The sensitivity of a newly designed staining protocol is usually estimated using 1D SDS-PAGE of serially diluted protein samples. However, this approach is not predictive and satisfactory for 2D SDS-PAGE capable of resolving hundreds or thousands of different proteins in a single analysis. In this work, a new fast staining protocol recently introduced by Dong et al. (PLoS One 2011, 6, e22394) was compared to colloidal CBS. The number of detectable spots in 2D SDS-PAGE of identical blood plasma samples in repeated runs was chosen as a sensitivity criterion. Further, the influence of gel boiling on the subsequent protein identification by MS was investigated. In spite of its advantages, the staining protocol according to Dong et al. (PLoS One 2011, 6, e22394) seems to be less sensitive than colloidal Coomassie staining when the number of detected spots is the evaluating criterion. No obvious influence of gel boiling on the protein identification was observed.
Heavy chain disease with monoclonal incomplete mu-heavy chains (mu-HCD) is a rare disorder usually associated with an underlying lymphoproliferative malignancy. Laboratory diagnosis of patients with mu-HCD is usually challenging and the monoclonal protein is not detected by electrophoresis in up to 75% of mu-HCD cases. We describe a patient with multiple malignancies in whom we detected and characterized monoclonal mu-heavy chains using immunofixation electrophoresis, capillary zone electrophoresis with immunotyping, and high resolution two-dimensional electrophoresis. The high resolution 2D electrophoresis enabled us to determine the molecular weight of the mu-heavy chains. The abnormal protein concentration in the serum was unusually high, 38 g/l measured in our patient is the highest reported value in the literature so far.
Two-dimensional differential gel electrophoresis (2-D DIGE) is a modification of the well-known twodimensional method (2-DE). The new method enables separation of two protein samples on one 2-D gel thus avoiding problems associated with reproducibility of 2-D gels and makes it possible to compare different samples. The only extra step in 2-D DIGE is sample labelling with cyanine fluorescent dyes (CyDyesTM). Two types of labelling are used: with succinimidyl esters and with maleimide derivatives of the dyes to label lysine and cysteine residues, respectively. For detection and quantification of protein spots in gels, a special fluorescent scanner or a CCD camera are required. From densitometric analysis of 2-D DIGE gels, sets of multidimensional data are obtained, which are then analysed by statistical methods. Protein spots can be cut from 2-D DIGE gels and further analysed by MS. The 2-D DIGE method affords much better quantification of proteins in samples. The separation of two samples at a time leads to a large reduction in the used amount of 2-D gels.
Application of Tris-N-[Tris(hydroxymethyl)methyl]glycine gels for 2DE is hampered by formation of mixed CHAPS-SDS micelles resulting in typical swirling pattern in the low mass range, which makes reliable quantitative and qualitative gel evaluation impossible. Modification of 2DE strip equilibration procedure prevented the direct interaction between both detergents during equilibration process, thus substantially improving gel separation.
Natural resistance to Mycobacterium bovis bacillus Calmette-Guerin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two-dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophages transfected with Nramp1-ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele-carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn-superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl-2 according to the immunoblot analysis. When the splenic macrophages carrying the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.
- MeSH
- 2D gelová elektroforéza * MeSH
- buněčné linie MeSH
- makrofágy * metabolismus MeSH
- membránové proteiny * genetika MeSH
- multivariační analýza MeSH
- Mycobacterium bovis * imunologie MeSH
- myši MeSH
- přirozená imunita MeSH
- proteiny přenášející kationty * MeSH
- transportní proteiny * genetika MeSH
- tuberkulóza MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- 2D gelová elektroforéza MeSH
- antigeny bakteriální biosyntéza izolace a purifikace MeSH
- bakteriální proteiny biosyntéza izolace a purifikace MeSH
- časové faktory MeSH
- Francisella tularensis imunologie metabolismus MeSH
- myši MeSH
- tularemie imunologie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
