Isolation and Analysis of Proteoglycans and Glycosaminoglycans from Archaeological Bones and Teeth
考古骨和牙齿中蛋白多糖和糖胺聚糖的分离及分析
发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2201 浏览次数: 7476
评审: Yanjie LiLi TangFilipa Vaz
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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 (蛋白聚糖)
Glycosaminoglycans (糖胺聚糖)
Archaeological bones (考古骨)
Archaeological teeth (考古牙齿)
Isolation (分离)
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
- Face masks, gloves and clean laboratory coats
- Safety glasses
- Autoclaved sandpaper (medium sheet - 100)
- Autoclaved cheesecloth
- Autoclaved A4 paper
- Autoclaved pipette tips
0.1-10 μl (Eppendorf, catalog number: 022491300 )
2-200 μl (Eppendorf, catalog number: 022491334 )
50-1,000 μl (Eppendorf, catalog number: 022491351 ) - DNA grade 15 ml tubes
- Amicon® Ultra-15 centrifugal filter unit (3K pore size) (EMD Millipore, catalog number: UFC900308 )
- Poly-Prep® chromatography column (Bio-Rad Laboratories, catalog number: 7311550 )
- 50 ml tubes
- Parafilm
- Guanidine hydrochloride (Sigma-Aldrich, catalog number: 177253 or G4505 )
Note: The product “ 177253 ” has been discontinued. - Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
- Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: RES20908-A7 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
- Autoclaved MilliQ water
- cOmplete proteinase inhibitor cocktail EDTA-free (Roche Diagnostics, catalog number: 05056489001 )
- Urea (Sigma-Aldrich, catalog number: U5378 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
- Sodium acetate (CH3COONa) (Sigma-Aldrich, catalog number: S2889 )
- Maxatase (Biocon Laboratories, São Paulo, Brazil)
- Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
- Methanol (Sigma-Aldrich, catalog number: 34860 )
- Standard chondroitin sulfate (extracted from shark cartilage) (Sigma-Aldrich, catalog number: C4384 )
- Standard dermatan sulfate (Sigma-Aldrich, catalog number: 1171455 )
- Standard heparan sulfate (extracted from bovine kidney) (Sigma-Aldrich, catalog number: H7640 )
- Standard hyaluronic acid (extracted from human umbilical cord) (Sigma-Aldrich, catalog number: H1751 )
Note: This product has been discontinued. - Low-mr agarose (Bio-Rad Laboratories, catalog number: 1620100 )
- Tris(hydroxymethyl)aminomethane (Tris) (Sigma-Aldrich, catalog number: 252859 )
- Tween 20 (Bio-Rad Laboratories, catalog number: 1706531 )
- EDTA III (Sigma-Aldrich, catalog number: EDFS )
- Sodium azide (Merck, catalog number: 106688 )
- Bovine serum albumin (Sigma-Aldrich, catalog number: A9418 )
- Sodium hypochlorite solution (see Recipes)
- Maxatase solution (see Recipes)
- Blocking buffer (see Recipes)
Equipment
- Pipettes
0.5-10 μl (Eppendorf, catalog number: 4920000024 )
2-20 μl (Eppendorf, catalog number: 4920000040 )
20-200 μl (Eppendorf, catalog number: 4920000067 )
100-1,000 μl (Eppendorf, catalog number: 4920000083 ) - Autoclave
- Drill (DRAPER TOOLS, catalog number: 79340 )
Note: It is used with sterilized mounted grinding stones (cylindrical and large cone shaped for bones, and tree and large cone shaped for teeth) (immersed in sodium hypochlorite solution for 15 min, dried and autoclaved). - Tube rotator
- SpeedVac concentrator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: SavantTM SC210 P1 SpeedVacTM , catalog number: SC210P1-115)
- Water bath
- Fume hood
- Ice maker
- Microcentrifuge, capable of reaching up to 16,000 x g
- Centrifuge, capable of reaching up to 2,000 x g
- Vortex mixer
- Laboratory oven
- Quick Scan 2000 densitometer (Helena Laboratories, model: QuickScan 2000 )
- Elisa ELX 800 Wallac Victor2 1420 Multilabel Counter (PerkinElmer, model: Elisa ELX 800 Wallac Victor2 1420 )
- Magnetic stirrer
Software
- Windows-based Quick Scan 2000 software (Helena Laboratories)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Coulson-Thomas, Y. M., Coulson-Thomas, V. J. and Norton, A. L. (2017). Isolation and Analysis of Proteoglycans and Glycosaminoglycans from Archaeological Bones and Teeth. Bio-protocol 7(7): e2201. DOI: 10.21769/BioProtoc.2201.
- Coulson-Thomas, Y. M., Coulson-Thomas, V. J., Norton, A. L., Gesteira, T. F., Cavalheiro, R. P., Meneghetti, M. C., Martins, J. R., Dixon, R. A. and Nader, H. B. (2015). The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth. PLoS One 10(6): e0131105.
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分类
生物化学 > 其它化合物 > 蛋白聚糖
生物化学 > 其它化合物 > 糖胺聚糖
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