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Two-dimensional electrophoresis (2DE) is a powerful method for separation of complex mixtures of proteins. The standard procedure is not, however, well suited to analysis of articular cartilage, which contains high concentrations of proteoglycans, the polyanionic glycosaminoglycan chains of which interfere with isoelectric focusing. We have developed a method for selective removal of proteoglycans by precipitation with cetylpyridinium chloride, after which the residual cartilage proteins are amenable to conventional 2DE analysis. Using this method, reproducible 2D-patterns can be obtained from proteins secreted by articular cartilage. The separated proteins may then be visualized by metabolic radiolabeling and silver staining, digested in gel with trypsin, and identified by tandem mass spectrometry.

Type

Journal article

Journal

Methods in molecular medicine

Publication Date

01/2007

Volume

136

Pages

349 - 359

Addresses

Kennedy Institute of Rheumatology Division, Imperial College London, UK.

Keywords

Cartilage, Articular, Chondrocytes, Humans, Arthritis, Rheumatoid, Osteoarthritis, Proteoglycans, Proteins, Proteome, Culture Media, Conditioned, Electrophoresis, Gel, Two-Dimensional, Tissue Culture Techniques