Electrophoresis and electroblotting of native collagens.
Electrophoretic and immunoblotting techniques, while now used routinely for the biochemical characterization of many proteins, have not been used for the identification of native collagens. We present here an acidic electrophoresis system using very low percentage acrylamide gels which maintains collagen solubility and allows migration of native dermal collagens. The method gives uniform gels which can be made mechanically stable for subsequent electroblotting. The resulting nitrocellulose transfer allows immunological detection of collagens using either polyclonal or monoclonal antibodies and can be used to screen antibody specificities. The majority of murine monoclonal antibodies directed against collagen bind only to conformational epitopes on the native triple-helical collagen, and thus cannot be screened by Western blotting. This method therefore enables the electrophoretic screening of these monoclonal antibodies and provides an alternative approach for their characterization.
Ramshaw
J A
JA
CSIRO, Division of Protein Chemistry, Parkville, Victoria, Australia.
Werkmeister
J A
JA
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002417:Cattle; D003094:Collagen / Q000032:analysis*; D003101:Collodion; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D011489:Protein Denaturation
D000911:Antibodies, Monoclonal; D003101:Collodion; D003094:Collagen