Abstract
We have developed methods for isolating proteoglycans and glycosaminoglycans from archaeological bones and teeth. These methods have been previously published (Coulson-Thomas et al., 2015) and are described here in more detail. In the case of glycosaminoglycans, the method was a previously described method (Nader et al., 1999) which we optimized for archeological samples.
Keywords: Proteoglycans, Glycosaminoglycans, Archaeological bones, Archaeological teeth, Isolation
Background
Bone tissue consists mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). As a result of binding tightly to hydroxyapatite, extracellular matrix proteins and PGs are protected from the destructive effects of temperature and chemical agents after death. However, to date only DNA and proteins had been successfully extracted from archaeological skeletons, and we therefore developed methods for isolating PGs and glycosaminoglycan (GAG) chains from archaeological bones and teeth. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease, and the analysis of these molecules from archaeological skeletons would unveil valuable paleontological information.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
If the GAGs are analyzed and quantified by agarose gel electrophoresis as described by Dietrich and Dietrich (1976), then the dried, stained agarose gels are scanned and quantified using a Quick Scan 2000 densitometer and software program (Helena Laboratories), following the suppliers instructions. The intensity of the sample GAG bands that appear on the gels is compared to standard bands of chondroitin sulfate, dermatan sulfate and heparan sulfate that result from a mixture of these GAGs of known concentration (all 1 mg/ml) applied to the same gels. The total quantity of GAGs is then calculated for 100 mg of bone or tooth powder, and this is then divided by the total quantity of protein extracted from 100 mg of bone or tooth powder to provide a GAG quantity in μg/mg of protein. If HA is quantified according to the probe-based sandwich ELISA assay described by Martins et al. (2003), then the plate is read using an Elisa ELX 800 Wallac Victor2 1420 Multilabel Counter (PerkinElmer) and the quantity of HA is calculated by comparison to standard HA added to the plate to produce a concentration curve (0, 0.48, 1.95, 7.8, 31.2, 125, 500 and 1,000 ng/ml HA). HA is then calculated per mg of protein to provide a quantity in ng/mg of protein.
Recipes
Acknowledgments
This protocol is from Coulson-Thomas et al., 2015. This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo). The archeological bones and teeth used in this work were provided by the University of Lincoln, Lincoln, UK.
References
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