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Fractionation Procedure (fractionation + procedure)
Selected AbstractsProteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionationELECTROPHORESIS, Issue 9 2009Alberto Acquadro Abstract Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana. [source] Whey-derived free fatty acids suppress the germination of Candida albicans in vitroFEMS YEAST RESEARCH, Issue 2 2007Martin Clément Abstract Bovine whey from the cheese-making industry contains several bioactive factors that promote health and prevent disease. Although many efforts have been made over the years to show that immunoglobulins, lactoperoxidase, lactoferrin, lysosyme and small peptides present in whey have antimicrobial activities against several pathogenic microorganisms, such activities have not been investigated so far for the lipid fraction of whey. Here, we have used an in vitro assay-based fractionation procedure to show that free fatty acids derived from whey cream specifically inhibit the germination of Candida albicans, a morphologic change associated with pathogenicity. Further fractionation by HPLC demonstrated that this activity can be mainly attributed to lauric acid, myristoleic acid, linoleic acid and arachidonic acid. [source] Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ionization target for mass spectrometric analysisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2006Johan Jacksén Abstract A CE separation of hydrophobic peptides followed by fractionation onto a prestructured MALDI target and off-line MS analysis was performed. An improved and partially automated manufacturing procedure of the previously described MALDI target is presented. This target is structurally coated with silicone and especially developed for hydrophobic peptides and proteins. Here, the target plate was designed specifically for the CE fraction collection. Different solvents were evaluated to meet the requirements of peptide solubility and compatibility to both the CE and MALDI methods and to the fractionation procedure. CE-MALDI-MS analysis of nine highly hydrophobic peptides from cyanogen bromide-digested bacteriorhodopsin is demonstrated. [source] A comparison of two methods for the isolation of free and occluded particulate organic matterJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 5 2005Angelika Kölbl Abstract Various methods exist for the isolation of particulate organic matter (POM), one of the soil-organic-matter (SOM) fractions reacting most sensitive on land-use or soil-management changes. A combination of density separation and ultrasonic treatment allows to isolate two types of POM: (1) free POM and (2) POM occluded in soil aggregates. POM fractions are closely linked to their biochemical function for the formation and stabilization of aggregates, therefore methods using different aggregate sizes may result in different POM fractions isolated. We evaluated two physical fractionation procedures to reveal whether they yield different POM fractions with respect to amount and composition, using grassland and arable soils with sandy-loam to sandy,clay-loam texture and thus low macroaggregate stability. Method I used air-dried aggregates of <2.0 mm size and a low-energy sonication for aggregate disruption, method II used field-moist aggregates <6.3 mm and a high-energy,sonication procedure for aggregate disruption. POM fractions were analyzed by elemental analysis (C, N) and CPMAS 13C-NMR spectroscopy. With both methods, about similar proportions of the SOM are isolated as free or occluded POM, respectively. The free- and occluded-POM fractions obtained with method I are also rather similar in C and N concentration and composition as shown by 13C-NMR spectroscopy. Method II isolates a free- and occluded-POM fraction with significantly different C and N concentrations. NMR spectra revealed significant differences in the chemical composition of both fractions from method II, with the occluded POM having lower amounts of O-alkyl C and higher amounts of aryl C and alkyl C than the free POM. Due to the use of larger, field-moist aggregates with minimized sample pretreatment, two distinctly different POM fractions are isolated with method II, likely to be more closely linked to their biochemical function for the formation and stabilization of aggregates. High-energy sonication as in method II also disrupts small microaggregates <63 µm and releases fine intraaggregate POM. This fraction seems to be a significant component of occluded POM, that allows a differentiation between free and occluded POM in sandy soils with significant microaggregation. It can be concluded, that microaggregation in arable soils with sandy texture is responsible for the storage of a more degraded occluded POM, that conversely supports the stabilization of fine microaggregates. Ein Vergleich zweier Methoden zur Isolierung von freier und okkludierter partikulärer organischer Substanz Partikuläres organisches Material (POM) wird im Hinblick auf die Landnutzung als sensitive Fraktion der organischen Bodensubstanz (SOM) angesehen, aber die unterschiedlichen Methoden seiner Isolierung erschweren den Vergleich zwischen verschiedenen Studien. Wir haben zwei physikalische Fraktionierungsmethoden ausgewertet, um zu zeigen, ob sie im Hinblick auf Menge und Zusammensetzung zu unterschiedlichen POM-Fraktionen führen. Hierfür wurden Proben von Grünland- und Ackerböden verwendet. Für Methode I wurden luftgetrocknete Aggregate der Größe <2 mm verwendet, zu deren Zerstörung eine Ultraschallbehandlung mit geringem Energieeintrag eingesetzt wurde. Für Methode II wurden feldfeuchte Aggregate der Größe <6.3 mm und eine Ultraschallbehandlung mit vergleichsweise hoher Energie zur Aggregatzerstörung herangezogen. Mit beiden Methoden konnten zwei POM-Gruppen gewonnen werden: (1) freies POM und (2) in Bodenaggregaten eingeschlossenes POM. Die POM-Fraktionen wurden mittels Elementanalyse (C, N) und CPMAS- 13C-NMR,Spektroskopie untersucht. Methode I zeigte im Hinblick auf Menge und Zusammensetzung nur sehr geringe Unterschiede zwischen freien und okkludierten POM-Fraktionen. Methode II isolierte freie und okkludierte POM-Fraktionen mit signifikant unterschiedlichen C- und N-Konzentrationen. Auch die NMR-Spektren zeigten Unterschiede in der chemischen Zusammensetzung der mit Methode II gewonnenen Fraktionen, die sich in signifikant geringeren O-Alkyl-C-Gehalten bei höheren Aryl-C- und Alkyl-C-Gehalten des okkludierten POM nachweisen ließen. Die Verwendung von größeren, feldfeuchten Aggregaten und die Minimierung der Probenvorbehandlung führt zu einer besseren Differenzierung beider POM-Fraktionen, die wahrscheinlich ihre biologische Funktion besser widerspiegelt. Zusätzlich führt eine Ultraschall-Behandlung mit hohem Energieeintrag zur Zerstörung von kleinen Mikroaggregaten <63 µm und damit zur Freisetzung von feinem Intraaggregat-POM. Diese Fraktion scheint in sandigen Böden mit niedriger Makroaggregat-Stabilität aussagekräftiger zwischen freier und okkludierter POM unterscheiden zu können. Folglich ist eine an das Bodenmaterial angepasste Probenvorbehandlung und Fraktionierungsmethode entscheidend, um eine präzise Charakterisierung der POM-Fraktionen zu gewährleisten. 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