Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot

Osteoarthritis and Cartilage
André StruglicsL S Lohmander

Abstract

To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity. Seven human aggrecan fragments (calibrators), purified by CsCl gradient centrifugation and identified by Western immunoblot of N- and C-terminals, were used to develop calculation models. The models were used for identification of unknown aggrecan fragments each having one of their N- or C-terminals identified. The calibrator molecular weights (Mw) from sodium dodecyl sulfate (SDS)-gels (m), the Mw of amino acids (a) and the Mw of their carbohydrate substitution (g) were expressed as K = m/(a+g), or as K = 1.085m/(a+g) when compensation for the G1 domain was required. Using these models together with average K-values, 12 out of the 17 immuno-detected aggrecan fragments were calculated to a known protease cleavage site, while five were identified to domain levels. With six neoepitope antibodies together with antibodies against the G1- and G3-domain it was possible to predict the identity of several proteolytic fragments from different regions within the aggrecan monomer.

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Citations

Mar 5, 2011·Nature Protocols·Heather StantonAmanda J Fosang
Sep 12, 2009·Matrix Biology : Journal of the International Society for Matrix Biology·André Struglics, Staffan Larsson
Dec 24, 2013·Osteoarthritis and Cartilage·S LarssonA Struglics
Jul 14, 2010·The Biochemical Journal·André Struglics, Maria Hansson
Sep 15, 2011·Biochimica Et Biophysica Acta·Heather StantonAmanda J Fosang
Jan 18, 2018·Glycobiology·Bruce Caterson, James Melrose
Oct 17, 2007·The Journal of Biological Chemistry·Mirna Z IlicChristopher J Handley

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