M. Jortikka et al., A HIGH-SENSITIVITY DOT-BLOT ASSAY FOR PROTEOGLYCANS BY CUPROLINIC BLUE PRECIPITATION, Connective tissue research, 29(4), 1993, pp. 263-272
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.