Proteoglycans (PGs) are present in the extracellular matrix and on the cell membrane. As they exhibit a high solubility with glycosaminoglycans (GAGs), they can be extracted simply with buffer or with mild chaotropic agents such as guanidinium hydrochloride (GuHCl) and urea. There are two useful methods of isolation of proteoglycans: Anion-exchange chromatography and a density gradient ultracentrifugation. The former includes steps of column chromatography usually with anion-exchange and gel filtration chromatography columns. Further purification requires affinity gel chromatography of a specific antibody. The latter applies to PGs with a number of GAG chains. In either method, factors for successful preparation are easy extractability and the amount of the PG to be isolated from tissues. This section presents examples of isolation of large PGs of the extracellular matrix. |
| Category | Glycosaminoglycans |
| Protocol Name | Preparation of proteoglycans from tissues |
Authors
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Watanabe, Hideto
Institute for Molecular Science of Medicine, Aichi Medical University
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| KeyWords |
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Reagents
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| ● |
4 M GuHCl
· Cesium chloride
· Urea
· DEAE-Sephacel (GE Healthcare, Little Chalfont, UK)
· Protease inhibitors (INHs, working concentrations are: 1 mM PMSF, 25 mM EDTA, 10 mM NEM)
· Stock solutions are:100 mM PMSF in absolute ethanol, 0. 5 M EDTA, 100 mM NEM
· Superose 6 HR10/30 (GE Healthcare, Little Chalfont, UK) |
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Instruments
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Ultracentrifuge and roter (> 40,000 rpm) |
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| Methods |
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1. |
Extraction of PGs from tissues |
| 1) |
Collect tissue and cut into thin slices or mince into small pieces with ~1 mm square. |
Comment 1
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| 2) |
Weigh the sample, and add 10 times (volume /weight) 4 M GuHCl, 50 mM Tris-HCl, pH 8.0, containing INHs. |
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| 4) |
Centrifuge at 4,000 × g for 30 min, collect supernate as 1st extract. |
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| 5) |
Add 5 volume of 4 M GuHCl, 50 mM Tris-HCl, pH 8.0, containing proteinase inhibitors to the precipitate, and extract for 12–16 h at 4°C. |
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| 6) |
Centrifuge at 4,000 × g for 30 min, collect supernate as 2nd extract. |
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| 7) |
Combine the 1st and 2nd extracts. |
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2. |
Anion-exchange chromatography |
| 1) |
Add two volumes of 95% ethanol, 1.3% potassium acetate. |
Comment 1
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| 2) |
Rest at 4°C overnight or at −20°C for 30 min–2 h. |
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| 3) |
Centrifuge at 10,000 × g for 30 min, and remove supernate. |
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| 4) |
Dissolve the precipitate with 50 mM Tris-HCl, pH 8.0, containing 7 M urea. |
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| 5) |
Apply the sample to a DEAE-Sephacel equilibrated with 50 mM Tris-HCl, pH 8.0, 7 M urea. |
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| 6) |
Wash with 50 mM Tris-HCl, pH 8.0, 0.2 M NaCl. |
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| 7) |
Elute with 50 mM Tris HCl, containing a higher concentration of NaCl (up to 2 M). |
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3. |
Gel filtration chromatography |
| 1) |
Collect the eluate of DEAE-Sephacel, containing PGs. |
Comment 1
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| 2) |
Apply the sample to a gel filtration column equilibrated with 50 mM TrisHCl, pH 8.0, 7 M urea. |
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4. |
Cesium chloride density gradient ultracentrifugation (associative) for isolation of aggrecan |
| 1) |
Dilute the extract (10× dilution) by adding nine volumes of 50 mM Tris-HCl, pH 8.0, containing INHs. |
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| 2) |
Add Cesium chloride to a density of ~1.6 g/mL and mix to dissolve well. |
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| 3) |
Transfer the solution to ultracentrifuge tubes, and ultracentrifuge at 40, 000 × g for 60–96 h at 12°C. |
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| 4) |
Insert a tube of peristaltic pump to the bottom of the centrifuge tube, and aspirate the solution, dividing into six or more fractions from the bottom. Fractions are termed A1, A2, and so on. |
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| 5) |
Collect the fractions (usually bottom fractions A1 and A2) containing aggrecan. |
Comment 1
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5. |
Cesium chloride density gradient ultracentrifugation (dissociative) for isolation of aggrecan |
| 1) |
Add GuHCl to the fractions containing aggrecan to a final concentration of 4 M |
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| 2) |
Add cesium chloride to a density of 1.6 g/mL and mix to dissolve well. |
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| 3) |
Transfer the solution to ultracentrifuge tubes, and ultracentrifuge at 40, 000 × g for 60–96 h at 12°C. |
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| 4) |
Insert a tube of peristaltic pump to the bottom of the centrifuge tube, and aspirate the solution, dividing into six or more fractions from the bottom. Fractions are termed D1, D2, and so on. |
Comment 0
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| 5) |
Collect the fractions (usually bottom fractions D1 and D2) containing aggrecan. |
Comment 0
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| Notes |
- GuHCl is useful for extraction of PGs in the extracellular matrix of tissues. For some tissues such as brain, PGs can easily be extracted by stirring in 50 M Tris-HCl, pH 7.5, 0.15 M NaCl.
- Ethanol precipitation is often performed to decrease the volume of the sample or to replace the solvent. To apply to the sample in 4 M GuHCl, a short period of resting is recommended. Leaving overnight at −20°C will co-precipitate GuHCl. For samples in 7 M urea, ethanol precipitation is not applicable, as a large amount of urea precipitates together with PGs.
- To monitor PGs in column chromatography, dot blot analysis is performed. Specific antibodies such as 1C6 for aggrecan, anti-CSbeta for mouse versican (Merck Millipore, Billerica, MA), 2B1 for human versican, 6B6 for decorin can be used.
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| Date of registration：2015-07-06 14:01:59 |
- Hascall, V. C., and Kimura, J. H. (1982) Isolation and characterization. Methods in Enzymol. 82 (Eds, Cummings L. and Fredelicksen, D. W.), 769–800 [PMID : 7200566]
- Yanagishita, M. (2001) Isolation of proteoglycans from cell cultures and Tissues. Methods Mol Biol. (Ed, Iozzo, R. V.) 3–8 [PMID : 11450240]
- Schmalfeldt, M, Dours-Zimmermann, M. T., Winterhalter, K. H., and Zimmermann, D. R. (1998) Versican V2 is a major extracellular matrix component of the mature bovine brain. J. Biol. Chem. 273, 15758–15764 [PMID : 9624174]
- Sakai, K., Kimata, K., Sato, T., Gotoh, M, Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) Chondroitin sulfate N-acetylgalactosaminyltransferase-1 plays a critical role in chondroitin sulfate synthesis in cartilage. J. Biol. Chem. 282, 4152–4161 [PMID : 17145758]
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