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Quantification of proteins in tracheobronchoalveolar lavage fluid by isoelectric focusing and scanning densitometry.
The aim of this study was to evaluate whether isoelectric focusing is a technique that is capable of quantifying a number of proteins simultaneously, in a single analysis of tracheobronchoalveolar lavage fluid. The aim included presentation of an animal model for such analysis after exposure to a pulmonary irritant. The studies were performed on tracheobroncoalveolar lavage fluid from rats. One-millimetre-thick agarose gels were used. The gel plates were stained with Coomassie Brilliant Blue R 250, which primarily stains proteins. Quantification of different protein bands was performed by laser scanning densitometry and computerized processing of the results. The effects of different steps in the analysis were examined. Recommendations for application of the method are proposed. An investigation was also made of the variance for the entire method, the variance attributable to isoelectric focusing and individual variation, for different protein bands. A light-microscopic examination for mechanical injury caused by tracheobronchoalveolar lavage was performed. The effects of perfusion of the pulmonary circulation with 0.9% sodium chloride solution during lavage was investigated. Application of the method in an experiment in which rats exposed to tobacco smoke were compared with rats not thus exposed showed that the method is powerful enough to show significances between these two groups. Corresponding results were obtained when rats, the pulmonary circulation of which was perfused as stated above, were compared with non-perfused rats. The experiments showed that isoelectric focusing combined with laser scanning as applied in this study, can be used for quantification of a system of proteins in tracheobronchoalveolar lavage fluid when different groups are to be compared.
The aim of this study was to evaluate whether isoelectric focusing is a technique that is capable of quantifying a number of proteins simultaneously, in a single analysis of tracheobronchoalveolar lavage fluid. The aim included presentation of an animal model for such analysis after exposure to a pulmonary irritant. The studies were performed on tracheobroncoalveolar lavage fluid from rats. One-millimetre-thick agarose gels were used. The gel plates were stained with Coomassie Brilliant Blue R 250, which primarily stains proteins. Quantification of different protein bands was performed by laser scanning densitometry and computerized processing of the results. The effects of different steps in the analysis were examined. Recommendations for application of the method are proposed. An investigation was also made of the variance for the entire method, the variance attributable to isoelectric focusing and individual variation, for different protein bands. A light-microscopic examination for mechanical injury caused by tracheobronchoalveolar lavage was performed. The effects of perfusion of the pulmonary circulation with 0.9% sodium chloride solution during lavage was investigated. Application of the method in an experiment in which rats exposed to tobacco smoke were compared with rats not thus exposed showed that the method is powerful enough to show significances between these two groups. Corresponding results were obtained when rats, the pulmonary circulation of which was perfused as stated above, were compared with non-perfused rats. The experiments showed that isoelectric focusing combined with laser scanning as applied in this study, can be used for quantification of a system of proteins in tracheobronchoalveolar lavage fluid when different groups are to be compared.