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

Struglics, André LU ; Larsson, Staffan LU and Lohmander, Stefan LU (2006) In Osteoarthritis and Cartilage 14(9). p.898-905
Abstract
Objective: To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity. Method. 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. Results: 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 = 1.085 m/(a + g) when compensation for the G1 domain was required. Using... (More)
Objective: To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity. Method. 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. Results: 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 = 1.085 m/(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. Conclusions: 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. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. (Less)
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author
; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
proteolytic fragments, calculation model, Western immunoblot, aggrecan
in
Osteoarthritis and Cartilage
volume
14
issue
9
pages
898 - 905
publisher
Elsevier
external identifiers
  • wos:000239898500008
  • scopus:33746513929
ISSN
1063-4584
DOI
10.1016/j.joca.2006.02.016
language
English
LU publication?
yes
id
c70f5946-f57a-4dae-8be5-290534acfe09 (old id 155768)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16635583&dopt=Abstract
date added to LUP
2016-04-01 12:20:00
date last changed
2020-01-12 09:38:42
@article{c70f5946-f57a-4dae-8be5-290534acfe09,
  abstract     = {Objective: To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity. Method. 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. Results: 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 = 1.085 m/(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. Conclusions: 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. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.},
  author       = {Struglics, André and Larsson, Staffan and Lohmander, Stefan},
  issn         = {1063-4584},
  language     = {eng},
  number       = {9},
  pages        = {898--905},
  publisher    = {Elsevier},
  series       = {Osteoarthritis and Cartilage},
  title        = {Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot.},
  url          = {http://dx.doi.org/10.1016/j.joca.2006.02.016},
  doi          = {10.1016/j.joca.2006.02.016},
  volume       = {14},
  year         = {2006},
}