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A high sensitivity dot-blot assay for proteoglycans by cuprolinic blue precipitation.
epartment of Anatomy, University of Kuopio, Kuopio, Finland.
epartment of Anatomy, University of Kuopio, Kuopio, Finland. (Chondrogenic and Osteogenic Differentiation Group)ORCID iD: 0000-0002-6181-9904
epartment of Anatomy, University of Kuopio, Kuopio, Finland.
epartment of Anatomy, University of Kuopio, Kuopio, Finland.
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1993 (English)In: Connective Tissue Research, ISSN 0300-8207, E-ISSN 1607-8438, Vol. 29, no 4, 263-272 p., 8269703Article in journal (Refereed) Published
Abstract [en]

A highly sensitive blot-assay was developed for glycosaminoglycans (GAGs) and proteoglycans (PGs) utilizing a precipitation reaction by a cationic dye Cuprolinic Blue. The precipitates were deposited into 1-2 mm2 spots on nitrocellulose membrane by using a 96-well filtration apparatus. The dried sheet was digitized by a flat bed scanner and the intensity of the dots was quantitated by an image analysis software. The working range for chondroitin sulfate was 10-300 ng. The response of various GAGs differed according to the number of anionic groups, both sulphate and carboxyl groups being able to bind the dye. The sensitivity of the assay was decreased by high concentrations of GuC, CsC and protein, but not by nonionic detergents, common buffers and 8 M urea. Contact exposure to autoradiography film enabled quantitation of 25-250 DPM, and 1-10 DPM, of 35SO4-radioactivity in precipitated PGs after overnight and 14 days' exposures, respectively.

Place, publisher, year, edition, pages
Informa Healthcare, 1993. Vol. 29, no 4, 263-272 p., 8269703
Keyword [en]
Proteoglycans, glycosaminoglycans, dot-blot assay, Cuprolinic acid, image analysis
National Category
Biochemistry and Molecular Biology
Research subject
Analytical Chemistry; Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-108497PubMedID: 8269703OAI: oai:DiVA.org:umu-108497DiVA: diva2:853219
Available from: 2015-09-11 Created: 2015-09-11 Last updated: 2017-12-04

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