Methods of isolation and purification of bovine and rat osteocalcin

Measurement of osteocalcin (a bone protein containing gamma-carboxyglutamic acid, GLA-P) in the biological liquids and tissues is of interest for studying the mechanisms of bone diseases. It is also helpful in making a diagnosis and in following up the patients with diseases in which the osseous system is affected. Methods have been worked out for isolation and purification of osteocalcin from bovine and rat femur in order to use the purified substance as a starting material for developing radioimmunoassay (RIA) systems for each of these osteocalcins. The work went through the following stages: the processing of the femur bone, preparation of the 200 microns bone powder, demineralization of the powder, and concentration of the bone extract, purification of the extract by molecular exclusion chromatography on Sephadex G-100 and repurification to the product by ion exchanger chromatography in NaCl linear gradient, on DEAE Sephadex A-25. The purity of the substance obtained was tested by disk electrophoresis in polyacrylamide gel. The bovine and rat osteocalcins obtained had the adequate purity for being used as RIA reagents.