Elsevier

Analytical Biochemistry

Volume 113, Issue 2, 15 May 1981, Pages 301-312
Analytical Biochemistry

Chromatographic and electrophoretic techniques
A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples

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Abstract

A method is described for the quantitation of the relative amounts of types I and III collagens in rabbit lung tissue. This involved (i) repeated homogenization in the presence of 2% sodium dodecyl sulfate and the production of an acetone dried powder, (ii) reaction with cyanogen bromide, (iii) polyacrylamide gel electrophoresis, and (iv) densitometric scanning of proteins stained by Coomassie blue R-250. Several features of this procedure were shown to offer advantages over methods previously employed. First, the sodium dodecyl sulfate solution was shown to remove the bulk of noncollagen proteins leaving an insoluble residue which could then be reacted with cyanogen bromide without further purification. Second, cyanogen bromide was shown to solubilize essentially all of the collagen in the residue leaving an insoluble pellet with an amino acid analysis similar to elastin. Finally, to facilitate accurate quantitation, types I and III collagen standards were included with each gel so that a standard curve of protein versus staining density could be constructed. This method is assessed to be simpler and more accurate than those employed previously for the quantitation of collagens and can be applied to small tissue samples (<100 mg) such as would be obtained by lung biopsy.

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    This work was supported by a grant from the Medical Research Council of Great Britain.

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    Peter Cockerill was supported by funds from W. B. Pharmaceuticals. Present Address: Department of Molecular and Cell Biology, Chester beatty Research Institute, Fulham Road, SW3 6JB, London.

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