A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300 000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above M(r) range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pK(a), as the buffering counterion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6-200 kDa M(r) range, with minimal difficulties in the post electrophoretic identification processes.
The Plasmodium falciparum food vacuole (FV) is a lysosome-like organelle where erythrocyte hemoglobin digestion occurs. It is a favorite target in the development of antimalarials. We have used a tandem mass spectrometry approach to investigate the proteome of an FV-enriched fraction and identified 116 proteins. The electron microscopy analysis and the Western blot data showed that the major component of the fraction was the FV and, as expected, the majority of previously known FV markers were recovered. Of particular interest, several proteins involved in vesicle-mediated trafficking were identified, which are likely to play a key role in FV biogenesis and/or FV protein trafficking. Recovery of parasite surface proteins lends support to the cytostomal pathway of hemoglobin ingestion as a FV trafficking route. We have identified 32 proteins described as hypothetical in the databases. This insight into FV protein content provides new clues towards understanding the biological function of this organelle in P. falciparum.
Several zwitterionic detergents differing in their polar heads, linker parts and hydrophobia tail were synthesized and evaluated for their efficiency in protein solubilizers for two-dimensional electrophoresis. A model system consisting of human red blood cell ghosts was used for this purpose. This study leads to the description of several new efficient detergents and allowed us to derive structural constraints for the design and synthesis of efficient detergents for two-dimensional electrophoresis. These constraints apply to the hydrophilic head (sulfobetaine but not carboxybetaine), to the hydrophobic tail (12 to 16 alkyl carbons long, linear alkyl or alkylaryl) and to the presence and nature of the linker between the hydrophilic head and hydrophobic tail.
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