Biomedical subjects

Robert Wildgruber

Publications and source records attributed to Robert Wildgruber.

3 recordsLinked to original sources

PubMed2005

Free-flow isoelectric focusing of proteins remaining in cell fragments following sonication of thyroid carcinoma cells.

The method of preparing protein mixtures for electrophoretic analysis of membrane-associated cell proteins was improved. By sonication, about one-half of the proteins of thyroid cells were released into the supernatant, while the other half preferentially comprising membrane proteins still remained in cell fragments, which could be sedimented by centrifugation. After sonication, even those proteins which remained in cell fragments, could completely be dissolved by free-flow isoelectric focusing media. They migrated through the free-flow electrophoresis chamber without forming precipitates. Because of these improvements, it was possible to show that the two thyroid cancer cell lines ML-1 and ONCO-DG1 express cytokeratin 8 at similar rates, but cytokeratins 7 and 18 differently. In addition, the presence of inorganic pyrophosphatase, tubulin-beta-5, and tubulin-beta-1 chains in human thyroid cells was proved for the first time.

Cell Line, Tumor↗

PubMed2004

In-gel digestion of proteins using a solid-phase extraction microplate.

We present a new procedure for in-gel digestion of proteins introducing a combination of two different 96-well microplates. The two plates have incorporated small capillaries with a length of 2.4 mm in each well, one of which has 75-microm-inner diameter capillaries, whereas the second plate has reversed-phase-type capillaries fixed to it. The initial steps of the in-gel digestion process, comprising destaining, reduction/alkylation, dehydration, and digestion, was carried out in the plate containing 75-microm capillaries. Capillaries containing C18 reversed-phase modified monolithic silica rods of a 200-microm diameter were used for the second plate in which extraction and cleanup of peptides were carried out. Peptides were eluted directly from the solid-phase extraction plate onto the MALDI sample support. The separation of the process into two plates led to increased process stability, without compromising sensitivity, i.e. peptide recovery, making it suitable for true high-throughput protein identification. The handling of proteinases could easily be optimized, and no restrictions were made on chosen pH range through the absence of the solid phase in the initial steps of the protocol. Efficient binding of peptides to the solid phase and subsequent direct elution onto the MALDI sample support led to sensitivities in the attomole range. Performance of the process was demonstrated with tryptic digests of proteins stained with colloidal coomassie blue, silver, and the fluorescent stain SYPRO Ruby.

Animals↗

PubMed2002

Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

An image based two-dimensional (2-D) reference map of very alkaline yeast cell proteins was established by using immobilized pH gradients (IPG) up to pH 12 (IPG 6-12, IPG 9-12 and IPG 10-12) for 2-D electrophoresis and by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting for spot identification. Up to now 106 proteins with theoretical isoelectric points up to pH 11.15 and molecular mass between 7.5 and 115 kDa were localized and identified. Additionally, due to the improved resolution of steady-state isoelectric focussing with IPGs, even low copy number proteins with codon bias below 0.02 were detected and identified.

Databases as Topic↗