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Free-flow isoelectric focusing of proteins remaining in cell fragments following sonication of thyroid carcinoma cells
Authors:Obermaier Christian  Jankowski Vera  Schmutzler Cornelia  Bauer Johann  Wildgruber Robert  Infanger Manfred  Köhrle Josef  Krause Eberhard  Weber Gerhard  Grimm Daniela
Affiliation:FFE Weber GmbH, Martinsried, Germany.
Abstract: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.
Keywords:Free‐flow electrophoresis  Inorganic pyrophosphatase  Isoelectric focusing  Membrane proteins  Thyroid cancer cells
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