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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   

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本实验方案简略版
PLOS ONE
Jun 2015

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

  1. Face masks, gloves and clean laboratory coats
  2. Safety glasses
  3. Autoclaved sandpaper (medium sheet - 100)
  4. Autoclaved cheesecloth
  5. Autoclaved A4 paper
  6. 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 )
  7. DNA grade 15 ml tubes
  8. Amicon® Ultra-15 centrifugal filter unit (3K pore size) (EMD Millipore, catalog number: UFC900308 )
  9. Poly-Prep® chromatography column (Bio-Rad Laboratories, catalog number: 7311550 )
  10. 50 ml tubes
  11. Parafilm
  12. Guanidine hydrochloride (Sigma-Aldrich, catalog number: 177253 or G4505 )
    Note: The product “ 177253 ” has been discontinued.
  13. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
  14. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: RES20908-A7 )
  15. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  16. Autoclaved MilliQ water
  17. cOmplete proteinase inhibitor cocktail EDTA-free (Roche Diagnostics, catalog number: 05056489001 )
  18. Urea (Sigma-Aldrich, catalog number: U5378 )
  19. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  20. Sodium acetate (CH3COONa) (Sigma-Aldrich, catalog number: S2889 )
  21. Maxatase (Biocon Laboratories, São Paulo, Brazil)
  22. Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
  23. Methanol (Sigma-Aldrich, catalog number: 34860 )
  24. Standard chondroitin sulfate (extracted from shark cartilage) (Sigma-Aldrich, catalog number: C4384 )
  25. Standard dermatan sulfate (Sigma-Aldrich, catalog number: 1171455 )
  26. Standard heparan sulfate (extracted from bovine kidney) (Sigma-Aldrich, catalog number: H7640 )
  27. Standard hyaluronic acid (extracted from human umbilical cord) (Sigma-Aldrich, catalog number: H1751 )
    Note: This product has been discontinued.
  28. Low-mr agarose (Bio-Rad Laboratories, catalog number: 1620100 )
  29. Tris(hydroxymethyl)aminomethane (Tris) (Sigma-Aldrich, catalog number: 252859 )
  30. Tween 20 (Bio-Rad Laboratories, catalog number: 1706531 )
  31. EDTA III (Sigma-Aldrich, catalog number: EDFS )
  32. Sodium azide (Merck, catalog number: 106688 )
  33. Bovine serum albumin (Sigma-Aldrich, catalog number: A9418 )
  34. Sodium hypochlorite solution (see Recipes)
  35. Maxatase solution (see Recipes)
  36. Blocking buffer (see Recipes)

Equipment

  1. 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 )
  2. Autoclave
  3. 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).
  4. Tube rotator
  5. SpeedVac concentrator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: SavantTM SC210 P1 SpeedVacTM , catalog number: SC210P1-115)
  6. Water bath
  7. Fume hood
  8. Ice maker
  9. Microcentrifuge, capable of reaching up to 16,000 x g
  10. Centrifuge, capable of reaching up to 2,000 x g
  11. Vortex mixer
  12. Laboratory oven
  13. Quick Scan 2000 densitometer (Helena Laboratories, model: QuickScan 2000 )
  14. Elisa ELX 800 Wallac Victor2 1420 Multilabel Counter (PerkinElmer, model: Elisa ELX 800 Wallac Victor2 1420 )
  15. Magnetic stirrer

Software

  1. Windows-based Quick Scan 2000 software (Helena Laboratories)

Procedure

  1. Bone and tooth preparation
    Note: The investigator must wear a face mask, gloves and clean laboratory coat to ensure no contamination of the samples. These must be changed between samples. The benchtop must be cleaned as described below between samples and new (cleaned and sterilized) tools used for each sample. Modern (control) samples should be handled in a different location on a different day to the archaeological samples.
    1. Clean the benchtop with sodium hypochlorite solution (prepared as described in Recipes).
    2. Clean the archaeological bones and teeth using sterilized water and autoclaved cheesecloth to remove dirt.
    3. Allow to air dry (on the benchtop).
    4. Clean the bone samples (but not teeth) using autoclaved sandpaper.
      Note: Since tooth enamel forms a hard, smooth surface, sandpaper doesn’t have much of an effect on it and dirt particles can be removed by simply using the autoclaved cheesecloth.
    5. Wipe the bones and teeth with sodium hypochlorite solution using autoclaved cheesecloth, making sure the samples are exposed to sodium hypochlorite for 15 min for sterilization.
    6. Allow to air dry (on the benchtop).
    7. Place the bone and tooth samples on autoclaved A4 paper.
    8. Wearing safety glasses, drill the bone and tooth samples using the draper drill and sterilized mounted grinding stones (previously immersed in sodium hypochlorite solution for 15 min, dried and autoclaved) until the necessary amount of bone or tooth powder has been collected in DNA grade 15 ml tubes (25-100 mg of bone or tooth powder/tube).
    9. Store the bone powder and tooth powder at -20 °C until further processing.
      Note: Approximately 25 mg of bone or tooth powder will provide one PG or GAG sample later on for agarose gel electrophoresis.

  2. Proteoglycan extraction
    Note: The investigator must wear a face mask, gloves and clean laboratory coat to ensure no contamination of the samples. Modern (control) samples should be handled in a different location on a different day to the archaeological samples.
    1. Suspend 25-100 mg of the bone or tooth powder prepared above in 4.5 ml of a solution of 4 M guanidine-HCl, 20 mM NaH2PO4, 30 mM Na2HPO4, 300 mM EDTA, pH 7.4, containing cOmplete proteinase inhibitor cocktail.
      Note: The solution to suspend the bone or tooth powder is prepared with MilliQ water and contains one tablet of cOmplete proteinase inhibitor cocktail added to 50 ml of solution, as per the manufacturer’s recommendations.
    2. Maintain under constant rotation at 4 °C for 24 h to keep the solution moving and prevent sedimentation of the bone or tooth powder (e.g., on a tube rotator in a cold room).
    3. Add 9 ml of a solution of 7 M urea, 0.3 M NaCl, 0.05 M CH3COONa, pH 6.5, containing cOmplete proteinase inhibitor cocktail.
      Note: The solution is prepared with MilliQ water and one tablet of cOmplete proteinase inhibitor cocktail added to 50 ml of solution, as per the manufacturer’s recommendations.
    4. Maintain under constant rotation at 4 °C for an additional 24 h to keep the solution moving and prevent sedimentation of the bone or tooth powder (e.g., on a tube rotator in a cold room).
    5. Filter the solution through a Poly-Prep® chromatography column to remove fine particles at 4 °C (e.g., in a cold room).
    6. Concentrate and desalinize the solution using an Amicon® Ultra-15 centrifugal filter unit, at 4 °C, to approximately 500 μl.
    7. Add cOmplete proteinase inhibitor cocktail (from stock solution 25x conc., prepared according to the manufacturer’s recommendations) and vacuum dry in a centrifugal evaporator (freeze dry).
    8. Store the crude protein/proteoglycan extract at -20 °C until further processing.
      Note: 25 mg of bone or tooth powder provides approximately 5 μg of PGs.

  3. Glycosaminoglycan extraction
    Note: The investigator must wear a face mask, gloves and clean laboratory coat to ensure no contamination of the samples. Modern (control) samples should be handled in a different location on a different day to the archaeological samples.
    1. Suspend 25-50 mg of the bone or tooth powder prepared above in 1 ml of maxatase solution (prepared as described in Recipes).
    2. Incubate the samples in a water bath at 60 °C for 2 days.
    3. Add 0.1 sample volume of trichloroacetic acid (TCA), vortex and keep the samples on ice for 15 min.
      Note: TCA should be added using a glass pipet in a fume hood to avoid inhalation of vapours. When handling TCA avoid skin contact, wear safety glasses to protect eyes and wear gloves, preferably nitrile gloves. Always add TCA to the sample and not the reverse.
    4. Centrifuge the samples (15 min, 2,250 x g, at 4 °C) and collect the supernatant (placing it on ice).
    5. Slowly add 2 sample volumes of methanol in a dropwise manner whilst vortexing.
      Note: The complete volume of methanol must be added with constant vortexing in an uninterrupted manner.
    6. Allow the samples to sit at -20 °C overnight for GAGs to precipitate.
      Note: It is important the samples are handled with care (transported on ice and with no brisk movements) while transported to and from the freezer.
    7. Centrifuge the samples (10 min, 2,250 x g, at 4 °C).
    8. Discard the supernatant by pipetting and dry the pellet at room temperature (until dry) or in a laboratory oven (50 °C for approximately 1 h).
      Note: Remove the lid from the tubes and cover the opening with Parafilm. Pierce the Parafilm to allow the methanol to evaporate.
    9. Store the crude glycosaminoglycan extract at -20 °C until further processing.
      Note: 25 mg of bone or tooth powder provides approximately 5 μg of GAGs.

  4. PG and GAG analysis
    1. For analysis and quantification of PGs and GAGs (chondroitin sulphate, dermatan sulphate and heparan sulphate) by agarose gel electrophoresis as described by Dietrich and Dietrich (1976), suspend (1 μg/μl) the crude protein/PG extract and GAG extract in MilliQ water with or without cOmplete proteinase inhibitor cocktail (from stock solution 25x conc., prepared according to the manufacturer’s recommendations), respectively, and apply 5 μl to the agarose gel.
    2. For quantification of hyaluronic acid (HA) as described by Martins et al. (2003), suspend 20 μg of the GAG extract in 300 μl of blocking buffer (prepared as described in Recipes) for performing the assay in triplicate.

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

  1. Sodium hypochlorite solution (final concentration of 1.25%)
    Dilute laboratory bleach (sodium hypochlorite) 1:10 in autoclaved MilliQ water
    Use within 30 days
  2. Maxatase solution ([4 mg/ml] in 0.05 M Tris-HCl buffer [prepared with MilliQ water], pH 8.0, containing 1 M NaCl)
    For a final volume of 100 ml:
    1. Weigh 400 mg of maxatase and 5.84 mg of NaCl
    2. Add the maxatase and NaCl to 0.05 M Tris-HCl buffer, pH 8.0, and mix using a magnetic stirrer
    3. Pour into two 50 ml tubes and centrifuge for 30 min to 1 h at maximum speed
    4. Transfer the supernatant to 15 ml tubes and discard the white precipitate
    5. Store at -20 °C
  3. Blocking buffer (pH 7.75)
    0.05 M Tris-HCl
    0.15 M NaCl
    0.05% Tween 20
    0.02 mM EDTA III
    7.7 mM sodium azide
    1% bovine serum albumin

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

  1. 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.
  2. Dietrich, C. P. and Dietrich, S. M. (1976). Electrophoretic behaviour of acidic mucopolysaccharides in diamine buffers. Anal Biochem 70(2): 645-647.
  3. Martins, J. R., Passerotti, C. C., Maciel, R. M., Sampaio, L. O., Dietrich, C. P. and Nader, H. B. (2003). Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients. Anal Biochem 319(1): 65-72.
  4. Nader, H. B., Chavante, S. F., dos-Santos, E. A., Oliveira, T. W., de-Paiva, J. F., Jeronimo, S. M., Medeiros, G. F., de-Abreu, L. R., Leite, E. L., de-Sousa-Filho, J. F., Castro, R. A., Toma, L., Tersariol, I. L., Porcionatto, M. A. and Dietrich, C. P. (1999). Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates? Braz J Med Biol Res 32(5): 529-538.

简介

我们开发了从考古学骨骼和牙齿中分离蛋白聚糖和糖胺聚糖的方法。 以前已经公开了这些方法(Coulson-Thomas等人,2015),并且在这里更详细地描述。 在糖胺聚糖的情况下,该方法是先前描述的方法(Nader等人,1999),其针对考古样品进行了优化。
【背景】骨组织主要由矿物成分(羟基磷灰石)和由胶原,非胶原蛋白和蛋白聚糖(PG)组成的有机基质组成。 由于与羟基磷灰石紧密结合,细胞外基质蛋白和PG被保护免受死亡后温度和化学药剂的破坏作用。 然而,到目前为止,只有DNA和蛋白质已经从考古学骨骼中成功提取,因此我们开发了从考古学骨骼和牙齿中分离PG和糖胺聚糖(GAG)链的方法。 PGs和GAG在骨形态发生,体内平衡和退行性骨病中起主要作用,从考古骨骼分析这些分子将揭示有价值的古生物学信息。

关键字:蛋白聚糖, 糖胺聚糖, 考古骨, 考古牙齿, 分离

材料和试剂

  1. 面罩,手套和干净的实验室外套
  2. 安全眼镜
  3. 高压灭菌砂纸(中片 - 100)
  4. 高压灭菌的干酪布
  5. 高压灭菌A4纸
  6. 高压灭菌移液器技巧
    0.1-10μl(Eppendorf,目录号:022491300)
    2-200μl(Eppendorf,目录号:022491334)
    50-1,000μl(Eppendorf,目录号:022491351)
  7. DNA级别为15毫升的管子
  8. Amicon ®超级离心过滤器(3K孔径)(EMD Millipore,目录号:UFC900308)
  9. Poly-Prep ®色谱柱(Bio-Rad Laboratories,目录号:7311550)
  10. 50ml管
  11. 石蜡膜
  12. 胍盐酸盐(Sigma-Aldrich,目录号:177253或G4505)
    注意:产品"177253"已经停产。
  13. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S8282)
  14. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:RES20908-A7)
  15. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  16. 高压灭菌的MilliQ水
  17. 不含蛋白酶的蛋白酶抑制剂鸡尾酒(Roche Diagnostics,目录号:05056489001)
  18. 尿素(Sigma-Aldrich,目录号:U5378)
  19. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  20. 乙酸钠(CH 3 COONa)(Sigma-Aldrich,目录号:S2889)
  21. Maxatase(Biocon Laboratories,SãoPaulo,Brazil)
  22. 三氯乙酸(Sigma-Aldrich,目录号:T6399)
  23. 甲醇(Sigma-Aldrich,目录号:34860)
  24. 标准硫酸软骨素(从鲨鱼软骨提取)(Sigma-Aldrich,目录号:C4384)
  25. 标准硫酸皮肤素(Sigma-Aldrich,目录号:1171455)
  26. 标准硫酸乙酰肝素(从牛肾提取)(Sigma-Aldrich,目录号:H7640)
  27. 标准透明质酸(从人脐带提取)(Sigma-Aldrich,目录号:H1751)
    注意:本产品已停产。
  28. 低密度琼脂糖(Bio-Rad Laboratories,目录号:1620100)
  29. 三(羟甲基)氨基甲烷(Tris)(Sigma-Aldrich,目录号:252859)
  30. 吐温20(Bio-Rad Laboratories,目录号:1706531)
  31. EDTA III(Sigma-Aldrich,目录号:EDFS)
  32. 叠氮化钠(Merck,目录号:106688)
  33. 牛血清白蛋白(Sigma-Aldrich,目录号:A9418)
  34. 次氯酸钠溶液(见配方)
  35. Maxatase解决方案(见配方)
  36. 阻塞缓冲区(见配方)

设备

  1. 移液器
    0.5-10μl(Eppendorf,目录号:4920000024)
    2-20μl(Eppendorf,目录号:4920000040)
    20-200μl(Eppendorf,目录号:4920000067)
    100-1,000μl(Eppendorf,目录号:4920000083)
  2. 高压灭菌器
  3. 钻(DRAPER TOOLS,目录号:79340)
    注意事项:使用无菌安装的磨石(圆柱形和大锥形骨头,树和大锥形牙齿)(浸入次氯酸钠溶液15分钟,干燥并高压灭菌)。
  4. 管旋转器
  5. SpeedVac集中器(例如,Thermo Fisher Scientific,Thermo Scientific TM ,型号:Savant TM SC210 P1 SpeedVac TM ,目录号:SC210P1-115)
  6. 水浴
  7. 通风柜
  8. 制冰机
  9. 微量离心机能够达到16,000 x g
  10. 离心机,能够达到2,000 x g
  11. 涡街搅拌机
  12. 实验室烤箱
  13. Quick Scan 2000密度计(Helena Laboratories,型号:QuickScan 2000)
  14. Elisa ELX 800 Wallac Victor2 1420 Multilabel计数器(PerkinElmer,型号:Elisa ELX 800 Wallac Victor2 1420)
  15. 磁力搅拌器

软件

  1. 基于Windows的Quick Scan 2000软件(Helena Laboratories)

程序

  1. 骨和牙齿准备
    注意:调查人员必须佩戴面罩,手套和干净的实验室外套,以确保不会污染样品。这些必须在样本之间更改。必须如下所述对样品和每个样品使用的新的(经过清洁和消毒的)工具进行清洁。现代(控制)样本应在与考古样本不同的一天的不同位置处理。
    1. 用次氯酸钠溶液清洗台面(按照食谱中所述进行准备)
    2. 使用无菌水清洁考古学骨骼和牙齿,然后用高压灭菌的干酪布去除污垢
    3. 允许风干(在台面上)。
    4. 使用高压灭菌砂纸清洁骨骼样本(但不是牙齿)。
      注意:由于牙釉质形成了坚硬,光滑的表面,砂纸对其没有太大的影响,并且可以通过使用高压灭菌的干酪布来除去污垢颗粒。
    5. 用次氯酸钠溶液用高压灭菌的干酪布擦拭骨骼和牙齿,确保样品暴露于次氯酸钠15分钟以进行灭菌。
    6. 允许风干(在台面上)。
    7. 将骨头和牙齿样品置于高压灭菌的A4纸上
    8. 佩戴安全眼镜,使用钻孔钻头和无菌安装的磨石(预先浸入次氯酸钠溶液15分钟,干燥并高压灭菌)钻骨头和牙齿样品,直到在DNA级别15中收集必要数量的骨或牙粉(25-100毫克骨或牙粉/管)。
    9. 将骨粉和牙粉放在-20°C直到进一步加工。
      注意:约25mg的骨或牙粉将稍后提供一个PG或GAG样品进行琼脂糖凝胶电泳。

  2. 蛋白多糖提取
    注意:调查人员必须佩戴面罩,手套和干净的实验室外套,以确保不会污染样品。现代(控制)样本应在与考古样本不同的一天的不同位置处理。
    1. 将25-100mg上述制备的骨或牙粉悬浮于4.5ml 4M胍-HCl,20mM NaH 2 PO 4,30mM Na 2含有完全蛋白酶抑制剂混合物的200mM HPO 4,300mM EDTA,pH 7.4。
      注意:根据制造商的建议,用MilliQ水制备悬浮骨或牙粉的溶液,并含有一片加入到50ml溶液中的完全蛋白酶抑制剂混合物。
    2. 在4℃恒定旋转24小时以保持溶液移动,并防止骨骼或牙齿粉末沉淀(例如在冷室中的管旋转器上)。
    3. 加入9ml含有完全蛋白酶抑制剂混合物的7M尿素,0.3M NaCl,0.05M CH 3 COONa,pH6.5的溶液。
      注意:根据制造商的建议,将溶液用MilliQ水制备,并将一片cOmplete蛋白酶抑制剂混合物加入到50ml溶液中。
    4. 在4℃下保持恒定旋转另外24小时以保持溶液移动并防止骨骼或牙齿粉末沉积(例如,在冷室中的管旋转器上)。 />
    5. 通过Poly-Prep 色谱柱过滤溶液,以在4℃(例如,在冷室中)除去细颗粒。
    6. 使用Amicon Supreme 15离心过滤器,在4℃下将溶液浓缩并脱盐至约500μl。
    7. 加入完全蛋白酶抑制剂混合物(从储备溶液25x浓度,根据制造商的建议制备),并在离心蒸发器中真空干燥(冷冻干燥)。
    8. 将粗蛋白质/蛋白多糖提取物储存在-20°C直到进一步处理 注意:25mg骨或牙粉提供约5μg的PG。

  3. 糖胺聚糖提取
    注意:调查人员必须佩戴面罩,手套和干净的实验室外套,以确保不会污染样品。现代(控制)样本应在与考古样本不同的一天的不同位置处理。
    1. 将25-50mg上述制备的骨或牙齿粉末悬浮于1ml最大值溶液(按照食谱中所述制备)。
    2. 将样品在60°C的水浴中孵育2天
    3. 加入0.1体积的三氯乙酸(TCA),涡旋并将样品保持在冰上15分钟 注意:TCA应在通风橱中使用玻璃移液器加入,以避免吸入蒸气。当处理TCA避免皮肤接触时,佩戴安全眼镜以保护眼睛并佩戴手套,最好是丁腈手套。始终将TCA添加到样品中,而不是相反。
    4. 在4℃离心样品(15分钟,2,250×g,4℃),收集上清液(放在冰上)。
    5. 慢慢地逐滴加入2个体积的甲醇,同时涡旋 注意:必须以不间断的方式,以恒定的涡流加入完整体积的甲醇。
    6. 允许样品在-20℃下放置过夜,使GAG沉淀 注意:在运送到冰箱或从冰箱运送时,请务必小心处理样品(在冰上运输,没有轻快的运动)。
    7. 在4℃离心样品(10分钟,2,250×g /次)。
    8. 通过移液去除上清液,并在室温(直到干燥)或实验室烘箱(50℃,约1小时)内干燥沉淀。
      注意:从管中取下盖子,用Parafilm盖住开口。打破石蜡膜,让甲醇蒸发。
    9. 将粗糖胺聚糖提取物储存在-20°C直至进一步处理 注意:25mg骨或牙粉提供约5μgGAG。

  4. PG和GAG分析
    1. 通过Dietrich和Dietrich(1976)所述的琼脂糖凝胶电泳对PGs和GAGs(硫酸软骨素,硫酸皮肤素和硫酸乙酰肝素)进行分析和定量,在MilliQ中悬浮(1μg/μl)粗蛋白/ PG提取物和GAG提取物具有或不具有完全蛋白酶抑制剂混合物的水(从储备溶液25x浓度,根据制造商的建议制备),并将5μl应用于琼脂糖凝胶。
    2. 用于如Martins等人所述定量透明质酸(HA)。 (2003)中,将300μg封闭缓冲液(如食谱中所述制备)中的20μgGAG提取物悬浮,一式三份进行测定。

数据分析

如Dietrich和Dietrich(1976)所述,如果通过琼脂糖凝胶电泳分析和定量GAG,则使用Quick Scan 2000密度计和软件程序(Helena Laboratories)按照供应商的说明扫描和定量干燥的染色的琼脂糖凝胶。将出现在凝胶上的样品GAG带的强度与由应用于相同凝胶的已知浓度(全部1mg / ml)的这些GAG的混合物的硫酸软骨素,硫酸皮肤素和硫酸乙酰肝素的标准条带进行比较。然后计算100mg骨粉或牙粉的GAG总量,然后除以100mg骨或牙粉提取的蛋白质总量,以提供μg/ mg蛋白质中的GAG量。
如果根据Martins等人描述的基于探针的夹心ELISA测定来定量HA。 (2003)中,然后使用Elisa ELX 800 Wallac Victor2 1420多标记计数器(PerkinElmer)读取板,并且通过与添加到板中的标准HA进行比较来计算HA的量以产生浓度曲线(0,0.48,1.95, 7.8,31.2,125,500和1,000ng / ml HA)。然后根据mg蛋白质计算HA,以提供ng / mg蛋白质的量。

食谱

  1. 次氯酸钠溶液(终浓度为1.25%)
    稀释实验室漂白剂(次氯酸钠)1:10在高压灭菌的MilliQ水中 在30天内使用
  2. 在含有1M NaCl的0.05M Tris-HCl缓冲液[用MilliQ水制备,pH8.0)中的最大值溶液([4mg / ml])
    最终体积为100ml:
    1. 称量400毫克maxatase和5.84毫克的NaCl
    2. 将maxatase和NaCl加入到0.05M Tris-HCl缓冲液(pH8.0)中,并使用磁力搅拌器
      进行混合
    3. 倒入两个50ml管中,并以最大速度从30分钟离心至1小时
    4. 将上清液转移到15 ml管中,弃去白色沉淀物
    5. 储存于-20°C
  3. 阻塞缓冲液(pH 7.75)
    0.05M Tris-HCl
    0.15 M NaCl
    0.05%吐温20
    0.02 mM EDTA III
    7.7mM叠氮化钠
    1%牛血清白蛋白

致谢

该协议来自Coulson-Thomas等人,2015年。这项工作得到CAPES(协调员协会),CNPq(Conselho Nacional de DesenvolvimentoCientíficoeTecnológico)和FAPESP(保加利亚圣保罗圣保罗圣保罗圣保罗教堂)。这项工作中使用的考古学骨骼和牙齿由林肯大学,英国林肯大学提供。

参考文献

  1. Coulson-Thomas,YM,Coulson-Thomas,VJ,Norton,AL,Gesteira,TF,Cavalheiro,RP,Meneghetti,MC,Martins,JR,Dixon,RA和Nader,HB(2015)。考古学人体骨骼和牙齿中蛋白多糖和糖胺聚糖的鉴定。 PLoS One 10(6):e0131105。
  2. Dietrich,CP和Dietrich,SM(1976)。电泳酸性粘多糖在二胺缓冲液中的作用。 Anal Biochem 70(2):645-647。
  3. Martins,JR,Passerotti,CC,Maciel,RM,Sampaio,LO,Dietrich,CP and Nader,HB(2003)。  通过对正常和肝硬化患者血清的新的基于非竞争性荧光的测定法实际测定透明质酸。 319 1):65-72。
  4. Nader,HB,Chavante,SF,dos-Santos,EA,Oliveira,TW,de-Paiva,JF,Jeronimo,SM,Medeiros,GF,de-Abreu,LR,Leite,EL,de-Sousa-Filho,JF, Castro,RA,Toma,L.,Tersariol,IL,Porcionatto,MA和Dietrich,CP(1999)。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Coulson-Thomas, Y. M., Coulson-Thomas, V. . 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.
  2. 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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