Ultrathin-layer horizontal high-resolution two-dimensional electrophoresis of legume seed proteins with intermediate protein staining

Summary Legume seed proteins extracted with urea-containing buffer are fractionated by high-resolution 2D-electrophoresis (urea-IEF x pore gradient SDS-PAGE), both dimensions in horizontal ultrathin-layer polyacrylamide gel slabs. High reproducibility is obtained, because the first dimension is performed in a slab gel, where a large number of protein samples are separated under identical conditions. The gel of the first dimension (IEF) is fixed, stained with Coomassie Brilliant Blue G 250, and destained before application to the second-dimension gel (SDS-PAGE). Prestaining of the focused proteins does not alter the protein pattern obtained after SDS electrophoresis. Thus, bands are made visible before separation in the second dimension, and the amount of Ampholine in the dye front is reduced during electrophoresis. The first-dimension gels can easily be stored in the destaining solution until they are run in the second dimension. As the proteins are fixed in the gel, there is no loss of proteins and defocusing of bands due to diffusion during the SDS-equilibration procedure. Loading of the dimensionstable gel strip onto the second dimension gel is a very easy operation, since the ultrathin gel strip adheres to a plastic foil and is simply laid into a moulded gel through. The gel strip is not imbedded with polymerizing acrylamide or agarose solution like in the conventional gel-rod-techniques, because a very good surface contact is obtained between the two flat gels.

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Ultradiinnschicht horizontale, hochauflösende Zweidimensional-Elektrophorese von Leguminosensamen-Proteinen mit Protein-Zwischenfärbung

Zusammenfassung Mit harnstoffhaltigen Tris-Glycin-Puffer extrahierte Leguminosensamen-Proteine werden mit der hochauflösenden 2D-Elektrophorese (Harnstoff-IEF x Gradientengel-SDS-Elektrophorese) aufgetrennt, wobei beide Dimensionen in horizontalen ultradünnen, auf Folie polymerisierten Polyacrylamid-Flachgelen durchgeführt werden. Die Flachgel-Focussierung (1. Dimension) erlaubt die Trennung einer großen Anzahl von Proteinproben unter identischen Bedingungen, wodurch die Reproduzierbarkeit von 2D-Elektrophoresen erheblich verbessert wird. Das Gel der ersten Dimension (IEF) wird mit Coomassie BB G 250 gefdrbt, bevor die einzelnen Gelstreifen für die zweite Dimension (Gradientengel-SDS-Elektrophorese) verwendet werden. Dies hat den Vorteil, daß die focussierten Proteine bereits vor dem zweiten Trennungsschritt sichtbar gemacht und zudem die Trägerampholyte in der Farbstoff Front bei der SDS-Elektrophorese stark vermindert werden. Die vorgefärbten Focussierungsgele können problemlos in der Entfärbelösung für die SDS-Gradientengel-Elektrophorese aufbewahrt werden. Der Proteinverlust und die Banden-diffusion bei der SDS-Aquilibrierung werden durch die Fixierung im Focussierungsgel verhindert. Das 2-D-Proteinmuster wird durch das Vorfärben der focussierten Proteine nicht verdndert. Die dimensionsstabilen, auf Folie polymerisierten Focussierungsstreifen lassen sich einfach handhaben und müssen nicht wie Rundgele an die zweite Dimension aufpolymerisiert werden. Der Gel-Gel-Kontakt der beiden Flachgele ist ohne weitere Hilfsmaßnahmen ausgezeichnet.

     

Automated microarray system for the simultaneous detection of antibiotics in milk

A parallel affinity sensor array (PASA) for the rapid automated analysis of 10 antibiotics in milk is presented, using multianalyte immunoassays with an indirect competitive ELISA format. Microscope glass slides modified with (3-glycidyloxypropyl)trimethoxysilane were used for the preparation of hapten microarrays. Protein conjugates of the haptens were immobilized as spots on disposable chips, which were processed in a flow cell. Monoclonal antibodies against penicillin G, cloxacillin, cephapirin, sulfadiazine, sulfamethazine, streptomycin, gentamicin, neomycin, erythromycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with horseradish peroxidase generating enhanced chemiluminescence, which was recorded with a sensitive CCD camera. All liquid handling and sample processing was fully automated, and one analysis was carried out in milk within less than 5 min. The detection limits ranged from 0.12 (cephapirin) to 32 microg/L (neomycin). Penicillin G could be detected at the maximum residue limit (MRL); the detection limits for all other analytes were far below the respective MRLs. The PASA system proved to be the first immunochemical biosensor platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of milk in the dairy industry.     

Reflective and reflexive action control in patients with frontal brain lesions.

Two types of action control derived from the model of action phases (H. Heckhausen & P. M. Gollwitzer, 1987) were analyzed in patients with frontal lesions, patients with nonfrontal lesions, and university students. In Study 1, reflective action control in terms of goal selection was assessed, and impaired deliberation was found in patients with frontal lesions. Study 2 assessed reflexive action control in terms of automatic action initiation as a result of forming implementation intentions (P. M. Gollwitzer, 1999). All participants sped up their responses to critical stimuli by forming implementation intentions. Moreover, lesion patients with weak performances on the Tower of Hanoi (TOH) task did worse than patients with strong TOH performances in Study 1 but better than control participants in Study 2. Findings are interpreted as a functional dissociation between conscious reflective action control and automatic reflexive action control. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Implementation intentions and efficient action initiation.

Implementation intentions ("If I encounter Situation X, then I'll perform Behavior Y!") are postulated to instigate automatic action initiation (P. M. Gollwitzer, 1993, 1999). In 4 studies, the hypothesis was tested that implementation intentions lead to immediate action initiation once the specified situation is encountered, even under conditions of high cognitive load. First, individuals whose action control is known to be hampered by disruptive cognitive business, such as opiate addicts under withdrawal (Study 1) and schizophrenic patients (Study 2), benefited from forming implementation intentions. Second, the beneficial effect of implementation intentions was also found in 2 experiments with university students (Studies 3 and 4) in which cognitive load was experimentally induced by using dual task paradigms. Results of the 4 studies suggest that forming implementation intentions instigates immediate action initiation that is also efficient. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Development and application of oligonucleotide probes for in situ detection of thermotolerant Campylobacter in chicken faecal and liver samples

Based on Campylobacter 16S- and 23S-rRNA sequence data oligonucleotide probes specific for thermotolerant campylobacters and for members of the genus Campylobacter have been developed. The 16S-rRNA-targeted probe CAMP 653, recommended for a comprehensive detection of members of the genus Campylobacter, specifically detected all Campylobacter strains used in this study. Detection of thermotolerant species has been achieved by the 23S-rRNA-targeted probe CAJECO1427. Optimal hybridisation conditions have been derived for both probes from melting profiles of fluorescence-labelled probe-target hybrids recorded in fluorescence in situ hybridisation experiments (FISH). The FISH assay was evaluated both by spiking poultry faecal samples with Campylobacter jejuni and by detecting Campylobacter in naturally colonized chickens. C. jejuni was reliably detected at levels of 10(6) cfu/g faeces after a 3- h enrichment step in Blood Preston Selective broth. Low level contaminations (相似文献   

Single-molecule switching with non-contact atomic force microscopy

We report upon controlled switching of a single 3,4,9,10-perylene tetracarboxylic diimide derivative molecule on a rutile TiO(2)(110) surface using a non-contact atomic force microscope at room temperature. After submonolayer deposition, the molecules adsorb tilted on the bridging oxygen row. Individual molecules can be manipulated by the atomic force microscope tip in a well-controlled manner. The molecules are switched from one side of the row to the other using a simple approach, taking benefit of the sample tilt and the topography of the titania substrate. From density functional theory investigations we obtain the adsorption energies of different positions of the molecule. These adsorption energies are in very good agreement with our experimental observations.