Two-dimensional electrophoresis of proteins secreted from articular cartilage.
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.
Hermansson
Monika
M
Kennedy Institute of Rheumatology Division, Imperial College London, UK.
Saklatvala
Jeremy
J
Wait
Robin
R
D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D002358:Cartilage, Articular / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D019902:Chondrocytes / Q000166:cytology / Q000502:physiology; D017077:Culture Media, Conditioned / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000032:analysis*; D011509:Proteoglycans / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D046509:Tissue Culture Techniques
D017077:Culture Media, Conditioned; D011506:Proteins; D011509:Proteoglycans; D020543:Proteome