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Molecular and Biochemical Assays of Cartilage Components

  • Caroline D. Hoemann
Part of the Methods in Molecular Medicine book series (MIMM, volume 101)

Abstract

The procedure described below is useful for extracting proteins, nucleic acids, and glycosaminoglycans from 5–40 mg of cartilage or tissue-engineered cartilage samples. This extraction method will generate samples compatible with Western blot, RNase protection, dimethyl methylene blue (DMB) assay for glycosaminoglycan, Hoechst DNA assay, and hydroxyproline assay. Most soluble matrix molecules can be extracted from pulverized samples using 4 M guanidine HCl, during a 30-min period of vortex agitation at 4°C. Shorter agitation times can give inadequate solubilization. The guanidine HCl-insoluble pellet must be re-extracted with guanidine thiocyanate buffer, to solubilize RNA additionally. The final insoluble pellet can be rinsed with ethanol and digested with papain, to quantify collagen content as well as other insoluble or crosslinked material. Samples between 1 and 5 mg may be directly digested with a small volume of papain buffer for DMB, hydroxyproline, and Hoechst DNA assays.

Key Words

Papain hydrogel L-trans-hydroxyproline collagen glycosaminoglycan deoxyribonucleic acid chondroitin sulfate C standard curve plate reader fluorimeter Hoechst 33258 pulverize guanidine hydrochloride guanidine thiocyanate cartilage. Extraction 

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Caroline D. Hoemann
    • 1
  1. 1.Department of Chemical EngineeringÉcole Polytechnique de MontréalMontréalCanada

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