Analysis of Aggrecan Glycanation by Western Blot in Cell Culture

Chiara Gramegna Tota; Alessandra Leone; Antonio Rossi; Chiara Paganini

doi:10.1007/978-1-0716-2946-8_11

Analysis of Aggrecan Glycanation by Western Blot in Cell Culture

Methods Mol Biol. 2023:2619:141-151. doi: 10.1007/978-1-0716-2946-8_11.

Authors

Chiara Gramegna Tota¹, Alessandra Leone^{1

2}, Antonio Rossi³, Chiara Paganini¹

Affiliations

¹ Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Pavia, Italy.
² University School for Advanced Studies Pavia, IUSS Pavia, Pavia, Italy.
³ Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Pavia, Italy. antrossi@unipv.it.

PMID: 36662468
DOI: 10.1007/978-1-0716-2946-8_11

Abstract

Several experimental protocols are available to study the synthesis and secretion of proteoglycans in health and diseases, but there are few methods to analyse the intracellular processing of these macromolecules. We report a western blot analysis on medium and cell layer of primary chondrocyte culture to determine the glycanation status of aggrecan. Using a specific antibody against the aggrecan core protein and digesting an aliquot of sample with chondroitinase ABC, it is possible to analyse the whole aggrecan macromolecule and the core protein in order to evaluate defects in aggrecan glycanation.

Keywords: Aggrecan; Cell processing; Chondrocyte; Glycosaminoglycan; Proteoglycan; Secretion; Western blot.

MeSH terms

Aggrecans
Blotting, Western
Cell Culture Techniques
Extracellular Matrix Proteins*
Lectins, C-Type
Proteoglycans* / metabolism

Substances

Aggrecans
Extracellular Matrix Proteins
Proteoglycans
Lectins, C-Type