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Aβ Extraction from Murine Brain Homogenates
鼠脑组织匀浆中的Aβ蛋白的提取   

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Abstract

This protocol details beta-amyloid (Aβ) extraction from transgenic murine brain homogenates. Specifically, mechanical homogenization of brain tissue and sequential extraction of both soluble and insoluble proteins are detailed. DEA extracts soluble proteins, such as Aβ isoforms and APP. Formic acid enables extraction of insoluble protein aggregates, such as Aβ isoforms associated with plaques. This procedure produces soluble and insoluble extracts that are amenable to analysis of Aβ species via western blotting and/or enzyme-linked immunosorbent assays (ELISAs), and these results help assess amyloidogenic burden in animals.

Keywords: Beta-amyloid(淀粉样蛋白), ELISA(ELISA), Extraction(提取), Abeta(淀粉状蛋白质), Murine(小鼠)

Materials and Reagents

  1. 5.0 ml open-top polyallomer ultracentrifuge tubes (or tubes capable of undergoing high-speed centrifugation) (Denville Scientific, catalog number: U5022 )
  2. Diethylamine (DEA) (≥ 99.5%) (Sigma-Aldrich, catalog number: 471216 )
  3. 95% formic acid (FA) (AMRESCO, catalog number: 0961 )
  4. 100 mM NaCl (store at room temperature)
  5. Tris base (Thermo Fisher Scientific, catalog numeber: BP152 )
  6. 0.5 M sodium phosphate dibasic (Na2HPO4) (AMRESCO, catalog numeber: 0348 )
  7. 0.05% sodium azide (NaN3) (Thermo Fisher Scientific, catalog numeber: S2271 )
  8. 250 mM sucrose (Thermo Fisher Scientific, catalog numeber: S5 )
  9. 0.5 mM Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate (EDTA) (Thermo Fisher Scientific, catalog numeber: S311 )
  10. 0.5 mM Ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) (Sigma Aldrich, catalog numeber: 03777 )
  11. Tris-hydrochloride (Tris-HCl)
  12. 0.4% DEA in 100 mM NaCl (see Recipes)
  13. 0.5 M Tris-HCl (pH 6.8) (see Recipes)
  14. Formic acid neutralization buffer (see Recipes)
  15. Tissue homogenization buffer (THB) (see Recipes)
  16. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 ) (see Recipes)

Equipment

  1. Beckman Coulter Optima L-90K Ultracentrifuge (used with an SW50.1 rotor)
  2. Ultrasonic sonicator (see Note 7, below) (Kontes, model: KT50 , catalog number: 12038 )

Procedure

Note: The following protocol has been used to extract Aβ from multiple mouse models of Alzheimer’s disease [please see Cramer et al. (2012) and Casali et al. (2015)]. The user may need to modify dilutions of the final extracted product depending on the particular application (e.g., ELISA and/or Western blotting). Our lab usually dilutes DEA and FA fractions for Aβ ELISAs at least five-fold to fall within our in-house ELISA detection limits. For Western blots of Aβ and modified APP fragments, we recommend 10 to 50 micrograms protein per well, and for more details about Western blotting using the DEA-soluble extraction, please see Morales-Corraliza et al. (2012).

  1. Mechanical homogenization
    1. Mechanically homogenize brain tissue (see Note 1) in 850 µl cold THB buffer containing fresh protease inhibitor cocktail on ice. If using flash-frozen brains, immediately homogenize. Freshly dissected brains may also be used. Homogenize thoroughly enough such that a homogenous mixture results.
    2. Aliquot 250 µl of homogenate into 1.5 ml Eppendorf tubes for DEA/FA extraction on ice (see Note 2). Proceed to DEA/FA extraction below. If not immediately extracting, flash freeze samples on dry ice and store at -80 °C (Note 3).

  2. DEA/FA Aβ extraction
    1. To 250 µl homogenate, add 250 µl (Note 4) 0.4% DEA and vortex rigorously until mixture appears homogenous.
    2. Transfer 500 µl of the homogenate/DEA sample to a tube capable of undergoing high-speed centrifugation.
    3. Using a swinging-bucket rotor (Note 5), perform a high-speed spin at 135,000 x g for 1 h at 4 °C.
    4. Remove 425 µl supernatant and neutralize with 42.5 µl 0.5 M Tris-HCl (pH 6.8). Vortex. Divide into 220 µl aliquots and flash-freeze on dry-ice. Store at -80 °C (Note 3). There will be a residual amount of soluble fraction remaining in the tube with the pellet that will not affect the downstream extraction-only remove 425 µl supernatant.
    5. Using the homogenate pellet that remains from step B4, add 125 µl cold formic acid (Note 6). Keep tubes on ice.
    6. Sonicate each sample on ice for 1 min continuously between output amplitude of 30-50 (Note 7). The pellet should dissolve after this amount of time. If not, sonicate until the pellet dissolves.
    7. Perform another high-speed spin at 109,000 x g for 1 h at 4 °C.
    8. Remove 105 µl sample, and add 1.895 ml of formic-acid neutralization buffer. Vortex and then divide into 2 x 1 ml aliquots and flash-freeze on dry-ice. Store at -80 °C (Note 3).
      Note: For the expected yield of DEA soluble extracts and FA fractions, our lab routinely obtains between 1.0 to 3.0 mg/ml protein and approximately 0.1 to 0.5 mg/ml protein respectively.

Notes

  1. Our lab uses one brain hemisphere with cerebellum and midbrain removed and flash frozen on dry ice. Our lab does not remove brain meninges upon harvesting of the tissue.
  2. If performing other assays on brain tissue homogenate, aliquot the remaining homogenate accordingly for downstream application (e.g., Western blotting; RNA extraction; etc.).
  3. Provided storage at -80 °C in a properly functioning freezer and the samples are stored in tight-capped tubes, our lab has routinely used samples 6-months post-collection.
  4. The amount of DEA to add to homogenate is 1:1 (volume/volume).
  5. Usage of a swinging-bucket, or carriage, is essential in performing efficient extraction.
  6. The formic acid must be cold (i.e., chilled to at least 4 °C) in order to precipitate insoluble proteins.
  7. Our lab uses a Kontes micro-ultrasonic (20 KHz frequency) cell disrupter rated at 50-Watts power, 120 volts, and 2 amperes.

Recipes

  1. 0.4% DEA solution
    200 µl DEA
    1 ml 5 M NaCl
    ddH2O to 50 ml
    Stored at room temperature and use within 3 months
  2. Formic acid neutralization buffer
    1 M Tris base
    0.5 M Na2HPO4
    0.05% NaN3
    Stored at room temperature and use within 3 months
  3. Tissue homogenization buffer (THB)
    2 mM Tris (pH 7.4)
    250 mM sucrose
    0.5 mM EDTA
    0.5 mM EGTA
    q.s. RNase-free H2O
    Stored at 4 °C and use within 3 months
    Note: “q.s.” means “quantity required”.
  4. 0.5 M Tris-HCl (pH 6.8)
    39.4 g tris-hydrochloride
    q.s. ddH2O
    Adjust pH to 6.8
    Stored at room temperature and use within three months
  5. Protease inhibitor cocktail
    Use at 1:100 dilution and make fresh with each homogenization (if your desired downstream application examines phosphorylated proteins, add phosphatase inhibitors in addition to protease inhibitor cocktail).

Acknowledgments

This work was supported by a grant from the National Institutes of Health, R41-AG048658.

References

  1. Casali, B. T., Corona, A. W., Mariani, M. M., Karlo, J. C., Ghosal, K. and Landreth, G. E. (2015). Omega-3 fatty acids augment the actions of nuclear receptor agonists in a mouse model of Alzheimer's disease. J Neurosci 35(24): 9173-9181.
  2. Cramer, P. E., Cirrito, J. R., Wesson, D. W., Lee, C. Y., Karlo, J. C., Zinn, A. E., Casali, B. T., Restivo, J. L., Goebel, W. D., James, M. J., Brunden, K. R., Wilson, D. A. and Landreth, G. E. (2012). ApoE-directed therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models. Science 335(6075): 1503-1506.
  3. Morales-Corraliza, J., Berger, J. D., Mazzella, M. J., Veeranna, Neubert, T. A., Ghiso, J., Rao, M. V., Staufenbiel, M., Nixon, R. A. and Mathews, P. M. (2012). Calpastatin modulates APP processing in the brains of beta-amyloid depositing but not wild-type mice. Neurobiol Aging 33(6): 1125 e1129-1118.

简介

该方案详述了从转基因鼠脑匀浆中提取β-淀粉样蛋白(Aβ)。 具体来说,详细说明脑组织的机械均质化和可溶性和不溶性蛋白质的顺序提取。 DEA提取可溶性蛋白质,如Aβ异构体和APP。 甲酸能够提取不溶性蛋白质聚集体,例如与斑块相关的Aβ同种型。 该方法产生可溶性和不溶性提取物,其适于通过western印迹和/或酶联免疫吸附测定(ELISA)分析Aβ物质,并且这些结果有助于评估动物的淀粉样变性负担。

关键字:淀粉样蛋白, ELISA, 提取, 淀粉状蛋白质, 小鼠

材料和试剂

  1. 5.0ml开顶聚合物超离心管(或能够进行高速离心的管)(Denville Scientific,目录号:U5022)
  2. 二乙胺(DEA)(≥99.5%)(Sigma-Aldrich,目录号:471216)
  3. 95%甲酸(FA)(AMRESCO,目录号:0961)
  4. 100mM NaCl(在室温下储存)
  5. Tris碱(Thermo Fisher Scientific,目录数:BP152)
  6. 0.5M磷酸氢二钠(Na 2 HPO 4)(AMRESCO,目录数:0348)
  7. 0.05%叠氮化钠(NaN 3)(Thermo Fisher Scientific,目录数:S2271)
  8. 250mM蔗糖(Thermo Fisher Scientific,目录数:S5)
  9. 0.5mM乙二胺四乙酸,二水合二钠(EDTA)(Thermo Fisher Scientific,目录数:S311)
  10. 0.5mM乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma Aldrich,目录数:03777)
  11. Tris-盐酸(Tris-HCl)
  12. 0.4%DEA在100mM NaCl中(参见配方)
  13. 0.5 M Tris-HCl(pH 6.8)(参见配方)
  14. 甲酸中和缓冲液(参见配方)
  15. 组织匀浆缓冲液(THB)(参见配方)
  16. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)(参见Recipes)

设备

  1. Beckman Coulter Optima L-90K超速离心机(与SW50.1转子一起使用)
  2. 超声波超声波仪(见下面的注7)(Kontes,型号:KT50和目录号:12038)

程序

注意:以下方案已经用于从阿尔茨海默氏病的多种小鼠模型中提取Aβ[参见Cramer等人, (2012)和Casali et al。 (2015)]。用户可能需要根据具体应用(例如ELISA和/或Western印迹)修改最终提取产物的稀释度。我们的实验室通常将AβELISA的DEA和FA级分稀释至少五倍,以符合我们内部ELISA检测限。对于Aβ和修饰的APP片段的蛋白质印迹,我们推荐每孔10至50微克蛋白质,并且关于使用DEA-可溶性提取物的Western印迹的更多细节,参见Morales-Corraliza等人。 (2012)。

  1. 机械均质化
    1. 机械均匀化脑组织(见注1)在850微升冷THB 缓冲液在冰上含有新鲜的蛋白酶抑制剂混合物。如果使用 快速冷冻的大脑,立即匀浆。新鲜解剖的大脑 也可以使用。均匀充分,使得均匀 混合结果。
    2. 等分250微升匀浆到1.5毫升 Eppendorf管在冰上进行DEA/FA提取(见注2)。继续 DEA/FA提取。如果没有立即提取,闪光冻结 样品置于干冰上并储存于-80℃(注3)。

  2. DEA/FAAβ提取
    1. 向250μl匀浆中,加入250μl(注4)0.4%DEA并剧烈涡旋,直到混合物呈均匀。
    2. 将500μl匀浆/DEA样品转移到能够进行高速离心的管中。
    3. 使用旋转桶转子(注5),在4℃下,以135,000in x g 进行高速旋转1小时。
    4. 取出425μl上清液,并用42.5μl0.5M Tris-HCl中和 ?(pH6.8)。涡流。分成220μl等分试样并快速冷冻 干冰。储存于-80℃(注3)。将有剩余金额 可溶性级分保留在具有不会沉淀的团粒的管中 影响下游提取 - 仅除去425μl上清液
    5. 使用从步骤B4剩余的匀浆沉淀,加入125μl冷甲酸(注6)。将管保持在冰上。
    6. 每个样品在冰上超声1分钟连续输出之间 振幅30-50(注7)。此后颗粒应溶解 多少时间。如果没有,声波处理直到颗粒溶解
    7. 在4℃下,以109,000 x g 进行另一次高速旋转1小时。
    8. 取出105μl样品,加入1.895 ml甲酸中和 ?缓冲。涡旋,然后分成2×1ml等分试样并快速冷冻 在干冰上。储存在-80°C(注3)。
      注意:对于预期收益率 的DEA可溶性提取物和FA级分,我们的实验室常规获得 1.0至3.0mg/ml蛋白质和约0.1至0.5mg/ml 蛋白质。

笔记

  1. 我们的实验室使用一个大脑半球与小脑和中脑去除和快速冷冻在干冰上。我们的实验室在收获组织时不会清除脑膜
  2. 如果对脑组织匀浆进行其它测定,则将剩余的匀浆相应地分装以用于下游应用(例如:Western印迹; RNA提取; 等)。
  3. 在正常工作的冰箱中储存在-80°C,样品储存在密封管中,我们的实验室常规使用收集后6个月的样品。
  4. 加入匀浆中的DEA的量为1:1(体积/体积)
  5. 使用摆动桶或滑架对于进行有效的提取至关重要。
  6. 甲酸必须是冷的(即,冷却至至少4℃)以沉淀不溶性蛋白质。
  7. 我们的实验室使用Kontes微超声波(20 KHz频率)细胞破碎器,额定功率为50瓦,120伏和2安培。

食谱

  1. 0.4%DEA溶液
    200μlDEA
    1ml 5M NaCl
    ddH 2 O至50ml
    储存在室温下,并在3个月内使用
  2. 甲酸中和缓冲液
    1 M Tris碱
    0.5M Na 2 HPO 4
    0.05%NaN 3> / 储存在室温下,并在3个月内使用
  3. 组织匀浆缓冲液(THB)
    2mM Tris(pH7.4) 250mM蔗糖 0.5mM EDTA 0.5mM EGTA 适量无RNase的H 2 O 2/b 储存在4°C,并在3个月内使用
    注意:"q.s."表示"需要数量"。
  4. 0.5M Tris-HCl(pH 6.8)
    39.4克三盐酸盐 适量ddH sub 2 O
    将pH调节至6.8
    储存在室温下,并在三个月内使用
  5. 蛋白酶抑制剂混合物
    使用1:100稀释,并使每个均质化(如果您所需的下游应用程序检查磷酸化蛋白,除蛋白酶抑制剂鸡尾酒之外添加磷酸酶抑制剂)新鲜。

致谢

这项工作得到了国立卫生研究院R41-AG048658的资助。

参考文献

  1. Casali,B.T.,Corona,A.W.,Mariani,M.M.,Karlo,J.C.,Ghosal,K.and Landreth,G.E。(2015)。 Omega-3脂肪酸增强了核受体激动剂在阿尔茨海默病小鼠模型中的作用。/a> J Neurosci 35(24):9173-9181。
  2. 这些结果表明,在一个实施方案中,本发明的方法可以用于制备本发明的化合物,所述化合物包括但不限于下列化合物:Cramer,PE,Cirrito,JR,Wesson,DW,Lee,CY,Karlo,JC,Zinn,AE,Casali,BT,Restivo,JL,Goebel,WD,James,MJ,Brunden,KR,Wilson,DAand Landreth, GE(2012年)。 ApoE指导的治疗可快速清除AD小鼠模型中的β-淀粉样蛋白和反向缺陷。 科学 335(6075):1503-1506。
  3. Morales-Corraliza,J.,Berger,J.D.,Mazzella,M.J.,Veeranna,Neubert,T.A.,Ghiso,J.,Rao,M.V.,Staufenbiel,M.,Nixon,R.A.and Mathews,P.M。 Calpastatin调节β淀粉样蛋白沉积大脑中的APP加工,但不调控野??生型小鼠。 a> Neurobiol Aging 33(6):1125 e1129-1118。
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Copyright: © 2016 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. Casali, B. T. and Landreth, G. E. (2016). Aβ Extraction from Murine Brain Homogenates. Bio-protocol 6(8): e1787. DOI: 10.21769/BioProtoc.1787.
  2. Casali, B. T., Corona, A. W., Mariani, M. M., Karlo, J. C., Ghosal, K. and Landreth, G. E. (2015). Omega-3 fatty acids augment the actions of nuclear receptor agonists in a mouse model of Alzheimer's disease. J Neurosci 35(24): 9173-9181.
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