A HIGH-SENSITIVITY DOT-BLOT ASSAY FOR PROTEOGLYCANS BY CUPROLINIC BLUE PRECIPITATION

A highly sensitive blot-assay was developed for glycosaminoglycans (GA Gs) and proteoglycans (PGs) utilizing a precipitation reaction by a ca tionic dye Cuprolinic Blue. The precipitates were deposited into 1-2 m m2 spots on nitrocellulose membrane by using a 96-well filtration appa ratus. The dried sheet was digitized by a flat bed scanner and the int ensity of the dots was quantitated by an image analysis software. The working range for chondroitin sulfate was 10-300 ng. The response of v arious GAGs differed according to the number of anionic groups, both s ulphate and carboxyl groups being able to bind the dye. The sensitivit y of the assay was decreased by high concentrations of GuC. CsC and pr otein, but not by nonionic detergents, common buffers and 8 M urea. Co ntact exposure to autoradiography film enabled quantitation of 25-250 DPM, and 1-10 DPM, of (SO4)-S-35-radioactivity in precipitated PGs aft er overnight and 14 days' exposures, respectively.