Abstract
Composite agarose-polyacrylamide gel electrophoresis (CAPAGE) in gels of 1.2% w/v polyacrylamide and 0.6% w/v agarose can be used to examine the heterogeneity of full-length native proteoglycan populations and their fragments in crude tissue extracts, and when used in conjunction with immunoblotting and specific antibodies to proteoglycan core protein and glycosaminoglycan, side chain epitopes can provide significant information on the level of proteoglycan polydispersity/heterogeneity and a number of proteoglycan populations present in tissue samples. This can be a technically difficult technique, but it reveals significant information on proteoglycans from small tissue samples not possible by any other separation methodology. Native full-length and proteoglycan fragments are examined in this technique something which cannot be done in the popular SDS-PAGE format unless the glycosaminoglycan side chains are first removed. Furthermore, since proteoglycans do not require renaturation from SDS–protein complexes, the proteoglycan populations separated by native electrophoresis are highly reactive with antibodies in immunoblotting procedures. Despite the massive sizes of proteoglycans, transfer conditions have been determined which provide close to quantitative transfer to nitrocellulose membranes without exceeding the binding capacity of such membranes, avoiding bleed-through of the transferred proteoglycans. Development of biotinylated hyaluronan and its application in an affinity blotting procedure has also yielded significant information on aggregatable proteoglycan populations separated by CAPAGE from a number of cartilages and vascular tissues in health and disease. While the CAPAGE system can be a technically demanding technique to master particularly in gel preparation, all other steps are straightforward, and the method yields invaluable information on proteoglycan populations extracted from connective tissues in health and disease that cannot be ascertained by any other technique. Further improvements in the detection of proteoglycan features with the development of novel bio-affinity probes or new antibody preparations are expected to further improve the utility of CAPAGE separation methodology.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Vesterberg O (1989) History of electrophoretic methods. J Chromatogr 480:3–19
Smith B (1994) SDS polyacrylamide gel electrophoresis of proteins. Methods Mol Biol 32:23–34
Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Backman L, Persson K (2018) The no-nonsens SDS-PAGE. Methods Mol Biol 1721:89–94
Gallagher S (2006) One-dimensional SDS gel electrophoresis of proteins. Curr Protoc Immunol 8:Unit 8.4
Matsumoto H, Haniu H, Komori N (2019) Determination of protein molecular weights on SDS-PAGE. Methods Mol Biol 1855:101–105
Thomas R, Kurien BT (2019) Ultrarapid sodium dodecyl sulfate polyacrylamide mini-gel electrophoresis. Methods Mol Biol 1855:491–494
Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22
Williams T, Combs JC, Thakur AP, Strobel HJ, Lynn BC (2006) A novel Bicine running buffer system for doubled sodium dodecyl sulfate – polyacrylamide gel electrophoresis of membrane proteins. Electrophoresis 27:2984–2995
Brunelle J, Green R (2014) One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE). Methods Enzymol 541:151–159
Lilley D (2000) Analysis of global conformation of branched RNA species using electrophoresis and fluorescence. Methods Enzymol 317:368–393
Stowell J, Tang TTL, Seidel M, Passmore LA (2021) Gel-based analysis of protein-nucleic acid interactions. Methods Mol Biol 2263:321–339
Woodson S, Koculi E (2009) Analysis of RNA folding by native polyacrylamide gel electrophoresis. Methods Enzymol 469:189–208
McDevitt C, Muir H (1971) Gel electrophoresis of proteoglycans and glycosaminoglycans on large-pore composite polyacrylamide-agarose gels. Anal Biochem 44:612–622
Heinegård D, Sommarin Y, Hedbom E, Wieslander J, Larsson B (1985) Assay of proteoglycan populations using agarose-polyacrylamide gel electrophoresis. Anal Biochem 151:41–48
Adams M, McDevitt CA, Ho A, Muir H (1986) Isolation and characterization of high-buoyant-density proteoglycans from semilunar menisci. J Bone Joint Surg Am 68:55–64
Varelas J, Zenarosa NR, Froelich CJ (1991) Agarose/polyacrylamide minislab gel electrophoresis of intact cartilage proteoglycans and their proteolytic degradation products. Anal Biochem 197:396–400
Melrose J, Numata Y, Ghosh P (1996) Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure. Electrophoresis 17:205–212
Melrose J, Little CB, Ghosh P (1998) Detection of aggregatable proteoglycan populations by affinity blotting using biotinylated hyaluronan. Anal Biochem 256:149–157
Melrose J (2001) Cartilage and smooth muscle cell proteoglycans detected by affinity blotting using biotinylated hyaluronan. Methods Mol Biol 171:53–66
Shu C, Melrose J (2018) The adolescent idiopathic scoliotic IVD displays advanced aggrecanolysis and a glycosaminoglycan composition similar to that of aged human and ovine IVDs. Eur Spine J 27:2102–2113
Ghosh P, Melrose J, Cole TC, Taylor T (1992) A comparison of the high buoyant density proteoglycans isolated from the intervertebral discs of chondrodystrophoid and non-chondrodystrophoid dogs. Matrix 12:148–155
Heinegård D (1977) Polydispersity of cartilage proteoglycans. Structural variations with size and buoyant density of the molecules. J Biol Chem 252:1980–1989
Melrose J, Ghosh P, Taylor TK (1994) Proteoglycan heterogeneity in the normal adult ovine intervertebral disc. Matrix Biol 14:61–75
Kosakai M, Yosizawa Z (1979) A partial modification of the carbazole method of bitter and Muir for quantitation of hexuronic acids. Anal Biochem 93:295–298
Farndale R, Buttle DJ, Barrett AJ (1986) Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 883:173–177
Wu M, Stockley PG, Martin WJ 2nd (2002) An improved Western blotting technique effectively reduces background. Electrophoresis 23:2373–2376
Caterson B, Christner JE, Baker JR, Couchman JR (1985) Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans. Fed Proc 44:386–393
Cheng H, Caterson B, Yamauchi M (1999) Identification and immunolocalization of chondroitin sulfate proteoglycans in tooth cementum. Connect Tissue Res 40:37–47
Christner J, Caterson B, Baker JR (1980) Immunological determinants of proteoglycans. Antibodies against the unsaturated oligosaccharide products of chondroitinase ABC-digested cartilage proteoglycans. J Biol Chem 255:7102–7105
Couchman J, Caterson B, Christner JE, Baker JR (1984) Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues. Nature 307:650–652
Caterson B, Christner JE, Baker JR (1983) Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan. J Biol Chem 258:8848–8854
Brown S, Melrose J, Caterson B, Roughley P, Eisenstein SM, Roberts S (2012) A comparative evaluation of the small leucine-rich proteoglycans of pathological human intervertebral discs. Eur Spine J Suppl 2:S154–S159
Melrose J, Fuller ES, Roughley PJ, Smith MM, Kerr B, Hughes CE, Caterson B, Little CB (2008) Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues. Arthritis Res Ther 10:R79
Young A, Smith MM, Smith SM, Cake MA, Ghosh P, Read RA, Melrose J, Sonnabend DH, Roughley PJ, Little CB (2005) Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis. Arthritis Res Ther 7:R852–RR86
Caterson B, Mahmoodian F, Sorrell JM, Hardingham TE, Bayliss MT, Carney SL, Ratcliffe A, Muir H (1990) Modulation of native chondroitin sulphate structure in tissue development and in disease. J Cell Sci 97:411–417
Caterson B (2012) Fell-Muir lecture: chondroitin sulphate glycosaminoglycans: fun for some and confusion for others. Int J Exp Pathol 93:1–10
Hayes A, Hughes CE, Ralphs JR, Caterson B (2011) Chondroitin sulphate sulphation motif expression in the ontogeny of the intervertebral disc. Eur Cell Mater 21:1–14
Hayes A, Hughes CE, Smith SM, Caterson B, Little CB, Melrose J (2016) The CS sulfation motifs 4C3, 7D4, 3B3[−]; and Perlecan identify stem cell populations and their niches, activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Stem Cells Dev 25:836–847
Hayes A, Smith SM, Caterson B, Melrose J (2018) Concise review: stem/progenitor cell proteoglycans decorated with 7-D-4, 4-C-3, and 3-B-3(−) chondroitin sulfate motifs are morphogenetic markers of tissue development. Stem Cells 36:1475–1486
Hayes A, Sugahara K, Farrugia B, Whitelock JM, Caterson B, Melrose J (2018) Biodiversity of CS-proteoglycan sulphation motifs: chemical messenger recognition modules with roles in information transfer, control of cellular behaviour and tissue morphogenesis. Biochem J 475:587–620
Melrose J, Isaacs MD, Smith SM, Hughes CE, Little CB, Caterson B, Hayes AJ (2012) Chondroitin sulphate and heparan sulphate sulphation motifs and their proteoglycans are involved in articular cartilage formation during human foetal knee joint development. Histochem Cell Biol 138:461–475
Shu C, Hughes C, Smith SM, Smith MM, Hayes A, Caterson B, Little CB, Melrose J (2013) The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc. Glycoconj J 30:717–725
Sorrell J, Mahmoodian F, Schafer IA, Davis B, Caterson B (1990) Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulfate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin. J Histochem Cytochem 38:393–402
Sorrell J, Carrino DA, Caplan AI (1993) Structural domains in chondroitin sulfate identified by anti-chondroitin sulfate monoclonal antibodies. Immunosequencing of chondroitin sulfates. Matrix 13:351–361
Visco D, Johnstone B, Hill MA, Jolly GA, Caterson B (1993) Immunohistochemical analysis of 3-B-(−) and 7-D-4 epitope expression in canine osteoarthritis. Arthritis Rheum 36:1718–1725
Dowdell J, Erwin M, Choma T, Vaccaro A, Iatridis J, Cho SK (2017) Intervertebral disk degeneration and repair. Neurosurgery 80:S46–S54
Fujii K, Yamazaki M, Kang JD, Risbud MV, Cho SK, Qureshi SA, Hecht AC, Iatridis JC (2019) Discogenic back pain: literature review of definition, diagnosis, and treatment. JBMR Plus 3:e10180
Siemionow K, An H, Masuda K, Andersson G, Cs-Szabo G (2011) The effects of age, sex, ethnicity, and spinal level on the rate of intervertebral disc degeneration: a review of 1712 intervertebral discs. Spine (Phila Pa 1976) 36:1333–1339
Teraguchi M, Yoshimura N, Hashizume H, Muraki S, Yamada H, Minamide A, Oka H, Ishimoto Y, Nagata K, Kagotani R, Takiguchi N, Akune T, Kawaguchi H, Nakamura K, Yoshida M (2014) Prevalence and distribution of intervertebral disc degeneration over the entire spine in a population-based cohort: the Wakayama spine study. Osteoarthr Cartil 22:104–110
Taylor T, Melrose J, Burkhardt D, Ghosh P, Claes LE, Kettler A, Wilke HJ (2000) Spinal biomechanics and aging are major determinants of the proteoglycan metabolism of intervertebral disc cells. Spine (Phila Pa 1976) 25:3014–3020
Melrose J, Roughley P, Knox S, Smith S, Lord M, Whitelock J (2006) The structure, location, and function of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages. J Biol Chem 281:36905–36914
Melrose J (2020) Perlecan, a modular instructive proteoglycan with diverse functional properties. Int J Biochem Cell Biol 128:105849
Whitelock J, Murdoch AD, Iozzo RV, Underwood PA (1996) The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. J Biol Chem 271:10079–10086
Disclosures
The author has received consultancy fees from Arthropharm and Sylvan Pharmaceuticals Pty Ltd. These companies had no input into the design, scope, or organization of this book chapter. The author has no conflicts to report. This study was funded by The Melrose Personal Research Fund, Sydney, Australia. JM was responsible for the writing of the chapter in its entirety.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Melrose, J. (2023). Separation and Identification of Native Proteoglycans by Composite Agarose-Polyacrylamide Gel Electrophoresis and Immunoblotting. In: Karamanos, N.K. (eds) Proteoglycans. Methods in Molecular Biology, vol 2619. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2946-8_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2946-8_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2945-1
Online ISBN: 978-1-0716-2946-8
eBook Packages: Springer Protocols