Special Considerations for Proteoglycans and Glycosaminoglycans and Their Purification

Jeffrey D. Esko1

1 University of Alabama, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.2
DOI:  10.1002/0471142727.mb1702s22
Online Posting Date:  May, 2001
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Abstract

In this unit, protocols describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. A support protocol presents a method for preparing serum from blood.

     
 
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Table of Contents

  • Basic Protocol 1: High‐Salt/Detergent Extraction of Proteoglycans and Glycosaminoglycans
  • Alternate Protocol 1: Alkali Extraction of Proteoglycans and Glycosaminoglycans
  • Basic Protocol 2: Anion‐Exchange Chromatography of Proteoglycans and Glycosaminoglycans
  • Alternate Protocol 2: CPC/Ethanol Precipitation of Proteoglycans and Glycosaminoglycans
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: High‐Salt/Detergent Extraction of Proteoglycans and Glycosaminoglycans

  Materials
  • Tissue sample, conditioned medium, biological fluids, or cultured cells
  • recipeGuanidine⋅HCl/Zwittergent 3‐12 extraction bufferor recipeTriton X‐100 extraction buffer, at 4°C
  • recipe200 × protease inhibitor stock solutions
  • Centrifuge and rotor (e.g., Sorvall SS‐34) or microcentrifuge, at 4°C
  • Additional reagents and equipment for measurement of uronic acids (unit 17.9) and metabolic radiolabeling (unit 17.4)

Alternate Protocol 1: Alkali Extraction of Proteoglycans and Glycosaminoglycans

  Additional Materials
  • 0.1 N or 10 N sodium hydroxide (NaOH; appendix 22)
  • recipe10 N acetic acid
  • Additional reagents and equipment for β‐elimination (unit 17.15)

Basic Protocol 2: Anion‐Exchange Chromatography of Proteoglycans and Glycosaminoglycans

  Materials
  • PG‐GAG extract (first protocol 1basic protocol or protocol 2alternate protocol)
  • recipeUrea/Zwittergent 3‐12 buffer
  • 20 mg/ml chondroitin sulfate or heparin in recipe20 mM Tris⋅Cl, pH 7.0
  • DEAE‐Sephacel (Pharmacia Biotech)
  • 0.2 M NaCl/ recipe50 mM sodium acetate, pH 6.0
  • recipeDEAE wash buffer
  • recipeDEAE elution buffer
  • Sephadex G‐25 (Pharmacia Biotech)
  • 10% ethanol
  • recipe20 mM Tris⋅Cl, pH 7.4 ( appendix 22)
  • Centrifuge and rotor (e.g., SS‐34) or microcentrifuge, at 4°C
  • Additional reagents and equipment for dialysis ( appendix 3A) and gel filtration and ion‐exchange chromatography (units 10.9 & 10.10)

Alternate Protocol 2: CPC/Ethanol Precipitation of Proteoglycans and Glycosaminoglycans

  Additional Materials
  • recipe20 mM Tris⋅Cl/ 0.2 M NaCl, pH 7.4
  • 5% (w/v) cetylpyridinium chloride hydrate (CPC; Aldrich), in water
  • recipe0.5 M sodium acetate ( appendix 22)
  • 95% ethanol, 4°C
  • recipe0.5 M sodium acetate in 10% (v/v) ethanol (sodium acetate/ethanol)
  • Additional reagents and equipment for ethanol precipitation (unit 2.1)
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Figures

Videos

Literature Cited

Literature Cited
   Bitter, T. and Muir, H.M. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4:330‐334.
   Filisetti‐Cozzi, T.M. and Carpita, N.C. 1991. Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197:157‐162.
   Hascall, V.C. and Kimura, J.H. 1982. Proteoglycans: Isolation and characterization. Methods Enzymol. 82:769‐800.
   Kimura, J.H., Caputo, C.B., and Hascall, V.C. 1981. The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma. J. Biol. Chem. 1256:4368‐4376.
   Kjellén, L. and Lindahl, U. 1991. Proteoglycans: Structure and interactions. Ann. Rev. Biochem. 60:443‐475.
   Parthasarathy, N. and Spiro, R.G. 1981. Characterization of the glycosaminoglycan component of the renal glomerular basement membrane and its relationship to the peptide portion. J. Biol. Chem. 256:507‐513.
   Rodén, L., Baker, J.R., Cifonelli, J.A., and Matthews, M.B. 1972. Isolation and characterization of connective tissue polysaccharides. Methods Enzymol. 28:73‐140.
   Sajdera, S.W. and Hascall, V.C. 1969. Protein polysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J. Biol. Chem. 244:77‐87.
   Yanagishita, M., Midura, R., and Hascall, V.C. 1987. Proteoglycans: Isolation and characterization from tissue cultures. Methods Enzymol. 138:279‐289.
Key References
   Rodén et al., 1972. See above.
  This review details conventional methods for isolating and characterizing the composition, size, and fine structure of GAG chains.
   Hascall et al., 1982. See above.
  This review describes detailed methods for purifying and characterizing tissue proteoglycans.
   Yanagishita et al., 1987. See above.
  This review extends accepted methodology for purifying and characterizing tissue proteoglycans to those found in cultured cells.
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