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Size-exclusion HPLC (size-exclusion + hplc)

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Chemistry	100%

Selected Abstracts

High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteins

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2006
José M. Rodriguez-Nogales
Abstract Methods for the analysis of maize proteins using HPLC and CE are reviewed. Most of the references cited in this review concern HPLC methods. Size-exclusion HPLC and especially RP-HPLC methods have been developed for characterization of normal and genetically modified maize, cultivar differentiation, and prediction of quality. Few CE methods for the analysis of maize proteins were found in the existing literature. Most of these methods focus on optimization of the separation of maize proteins using CZE and SDS-capillary gel electrophoresis. [source]

Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2005
Alexandru C. Lazar
Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Generation of catalytically active 6-phosphofructokinase from Saccharomyces cerevisiae in a cell-free system

FEBS JOURNAL, Issue 15 2000
Anke Edelmann
PFK1 and PFK2 coding for the subunits of 6-phosphofructokinase from Saccharomyces cerevisiae were cloned into plasmids suitable for runoff transcription. In vitro translation products of both kinds of subunit were obtained using rabbit reticulocyte lysate as the synthesis and folding system. They were monitored by chemiluminescent Western-blot analysis. Folding and assembly of the ,-subunit and ,-subunit of 6-phosphofructokinase were found to occur in the cell-free system resulting in an enzymatically active protein. The in vitro generated enzyme exhibits a folding state that is similar to that of the heterooctameric form of 6-phosphofructokinase in the presence of fructose 6-phosphate, ATP and ammonium sulfate, as demonstrated by size-exclusion HPLC followed by ELISA. [source]

Papain hydrolysates of casein: molecular weight profile and encapsulation in lipospheres

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2004
Cristiane MS Barbosa
Abstract Some reaction parameters were tested in the hydrolysis of casein by papain, in order to prepare hydrolysates with high oligopeptide contents, for either dietetic or pharmaceutical purposes. Five casein hydrolysates were prepared and then fractionated by size-exclusion HPLC. The rapid correct fraction area method was used for quantifying peptides and free amino acids. Among the five reaction conditions tested, three produced similar peptide profiles. However, the use of a temperature of 37°C and an E:S ratio of 2% is probably the most economical condition for use in large-scale manufacture. With the aim of masking the bitterness of these preparations, a new method, based on the encapsulation in lipospheres, was used. Also, second derivative spectrophotometry was used for the first time to measure the extent of encapsulation of protein hydrolysates, which changed from 50% to 83%. Moreover, the efficiency of this system was evaluated by analysing other parameters, which showed a reduction of hydrophobicity and bitterness of all samples, as well as good chemical stability during 60 days of storage under refrigeration. The electron microscopical analysis of liposheres showed an average size around 5.0 ± 1.0 µm. Copyright © 2004 Society of Chemical Industry [source]