Autoradiographic quantitation of radiolabeled proteoglycans

Radiolabeled proteoglycans or glycosaminoglycans were precipitated with the cationic dye safranin O onto a sheet of nitrocellulose filter using a dot-blot apparatus. An autoradiography film was exposed against the nitrocellulose sheet. The developed film and the nitrocellulose sheet were separately digitized in a flat-bed-gray-scale scanner connected to a microcomputer. An image analysis program of the microcomputer was used to quantify the density of the radioactivity dots produced in the film, and the intensity of the dye spots on nitrocellulose. With this procedure, a single sample containing the minimum of about 20 ng uronic acid and 5 dpm of incorporated 35SO4 was quantified for both total glycosaminoglycan content and radioactivity. Unincorporated 35SO4 and low molecular mass radioactivity (e.g. products of glycosaminoglycan degrading enzymes) did not interfere since they were quantitatively washed through the membrane before the assay.