Name: enrichment of detergent-insoluble protein aggregates from human postmortem brain
Uploaded: 2017-10-24T12:30:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

作者感谢 Drs 和利维, 埃默里神经病学系, 提供有用的意见和建议。这项工作的部分资金来自加速医学合作基金 (U01AG046161-02), 埃默里阿尔茨海默病研究中心 (P50AG025688) 和国家老龄补助金研究所 (R01AG053960-01) 运输这项研究也得到了部分的支持, 神经病核心的埃默里神经科学一核心设施补助金, P30NS055077。

道德声明: 所有脑组织都是从埃默里老年痴呆和 #39 的疾病研究中心 (自抗扰) 脑银行获得的。人类死后组织是在适当的机构审查委员会 (IRB) 协议下获得的. 
 1. 均匀化和分馏 <ol> <li> 组织选择 注意: 从健康控制 (Ctl) 和经病理证实的 AD 病例的冷冻死后额叶皮质组织从埃默里抗扰的脑银行 ( n = 2)。病理评价淀粉样菌斑的分布情况, 根据该协会建立一个老年痴呆症和 #39 的疾病的登记表. 半定量评分标准 24 , 虽然纤维缠结病理学根据 Braak 分期系统评估。 16 <ol> <li> 从健康控制 (Ctl) 和经病理证实的 AD 病例中获得冰冻死后的脑组织。利用干冰、钳子和刀片的容器, 在 tared 的一次性重量船上, 从每个组织样品中去除250毫克的灰质部分, 注意防止组织解冻。记录每个工件的重量, 使其均匀化. </li>
		<li> 用镊子和剃刀对灰色物质进行250毫克的目测检查, 找出外灰质与内白质之间的界面。避免白质, 更重要的是, 任何大型血管或血腥地区, 脑膜, 或蛛网膜。在切割过程中, 保持组织冷冻, 迅速工作, 并频繁地将称重的船与脑组织放在聚苯乙烯容器中干冰. </li>
		<li> 用分析天平记录每个冰冻切片的灰物质的确切重量。计算 dounce 均匀化所需的低盐 (LS) 缓冲器 (见表 1) 的体积 (5 毫升/克或20% 瓦特/伏). </li>
		<li> 准备10毫升 LS 缓冲区与1x 蛋白酶和磷酸酶抑制剂鸡尾酒和冰镇冰. </li>
		<li> 从干冰中取出称重的组织和称量的船, 并在解冻时将其切成大约 2 x 2 毫米的碎片。转到2毫升 pre-chilled dounce 均质管在冰上. </li>
		<li> 添加5毫升/克 (20% 瓦特/五) 的 ice-cold 低盐 (LS) 缓冲与1X 蛋白酶和磷酸酶抑制剂鸡尾酒. </li>
		<li> 质在冰上使用大约10招的高间隙杵 a 和15招低间隙杵 B. </li>
		<li> 使用玻璃巴斯德吸管将所有匀浆转移到2毫升聚丙烯管上。把管子和 #8220; #8221; (总匀浆), 组织病例数, 匀浆体积. 和 #160; 分0.8 毫升成一个标签1.5 毫升的管。任何多余的匀浆可以类似 aliquoted 和存储在-80 和 #176; C. </li>
		<li> 每0.8 毫升分, 添加100和 #181; L 每5米氯化钠和 10% (w/五) sarkosyl 的浓度分别为0.5 米和1% 瓦特/伏。通过反演和在冰上孵育15分钟的标签管与 #34; TH 和 #34; (总匀浆-Sarkosyl), 以指示匀是在 Sarkosyl 缓冲区 ( 表 1 ). </li>
		<li> 几种每管三 5 s 脉冲在冰上30% 振幅 (最大强度 = 40%) 使用 mircotip 探针. #160; </li> <li> 使用二辛可宁酸测定匀的蛋白质浓度 25 方法. 
		注: 每克组织5毫升的匀制备的平均蛋白质浓度为15-20 毫克/毫升。匀可以立即分馏或储存在-80 和 #176; C 直到使用. </li>
		<li> 使用 ice-cold 萨克缓冲区 ( 表 1 ) 将匀稀释至10毫克/毫升, 并配有1x 蛋白酶和磷酸酶抑制剂. </li>
		<li> 将每个 TH 匀浆的5毫克蛋白 (0.5 毫升) 转化为500和 #181; 聚碳酸酯 ultracentrifuge 管和对萨克缓冲器的配对平衡。将管装入 pre-chilled 转子, ultracentrifuge 在 18万 x g 处为30分钟, 在4和 #176; c. 将 sarkosyl 可溶性清 (S1) 转移到1.5 毫升管, 并在-80 和 #176 上储存; c. 
		注: (可选洗涤) 增加200和 #181; 萨克缓冲区 L 的 ultracentrifuge 管含有洗涤剂不溶性分数 (P1), 并从 ultracentrifuge 管底部取出小球 (P1) 使用200和 #181; l 吸管尖端. </li>
		<li> 简要脉冲-旋转2-3 秒 (和 #8804; 2500 x g ) 上的反转管, 离心将颗粒 (P1) 和缓冲器转移到下面的1.5 毫升管上。重的不溶性颗粒 (P1) 在萨克缓冲区通过移上下, 以确保颗粒被打乱. </li>
		<li> 对平衡和离心机在 18万 x g 为另外 30 min 在4和 #176; C. 和 #160; </li>
		<li> 丢弃可选洗涤上清液 (S2), 并在50-75 和 #181 中孵育 sarkosyl 不溶性颗粒 (P2); 尿素缓冲器 ( 表 1 ), 具有 1x PIC (蛋白酶和磷酸酶抑制剂鸡尾酒), 室温下30分钟, 溶解颗粒. 
		注: 温尿素缓冲液用于室温前, 避免 SDS 沉淀。对于洗涤剂敏感的应用, 从尿素缓冲液中省去 SDS, 考虑在尿素缓冲液溶液前, 在低盐缓冲液中洗涤不溶性颗粒 (P2). </li>
		<li> 将悬浮颗粒 (P2) 转换为0.5 毫升管, 并使用短 (1 s) mircotip 超声在20% 振幅 (最大强度 = 40%), 以完全溶解颗粒. </li>
		<li> 测定 sarkosyl 可溶性 (S1) 和不溶性 (P2) 组分的蛋白质浓度。使用这些分数立即或存储在-80 和 #176; C 直到使用. </li>
	</ol> </li> </ol> 2。免疫 <ol> <li> 西方印迹 注: 根据先前报告的程序进行了轻微修改, 进行了印迹。5 , 10 <ol> <li> 准备40和 #181; g 样品中总匀 (TH S), 萨克可溶性 (S1) 和沙克不溶 (P2) 分数在 1X Laemmli SDS 页样本缓冲区与5毫米 TCEP pH 7。在95和 #176 孵育样品; C 为 5 min. </li>
		<li> 在预制10井4-12% 双-三页凝胶中装载样品。Electrophorese 在 80 v 为第一厘米 (10 分钟) 和 120 v 为剩下的人 (60 分钟) 或直到跟踪染料到达胶凝体的底部. </li>
		<li> 使用新的刀片和凝胶刀来拆分-打开预制凝胶盒。轻轻地把梳子和底部的凝胶与一个新鲜的剃须刀刀片. </li>
		<li> 重200和 #181 中的不溶性球团 (P1); 移上下。
		<li> 在印迹机上使用干式传输方法将0.2 和 #181; m 硝化纤维素膜 (见材料表). </li>
		<li> 阻塞缓冲膜 (bb), 不吐温20为30分钟, 后跟 bb 与0.05% 吐温 20 (bb/T) 为30分钟。阻塞后, 用 TBS/T (0.1% 吐温 20) 冲洗膜, 5 分钟清除多余的阻塞缓冲区. </li>
		<li> 准备 1:1, 000 稀释主要抗体 pT231, Tau-2 (盘头), AT8和 EEA1 在 BB/T (0.05% 吐温 20). </li>
		<li> 在4和 #176 的一夜之间在主抗体溶液中孵育膜, 循环搅拌。在 TBS/吨冲洗膜 3 x 15 min. </li>
		<li> 用1:20,000 稀释的荧光标记近红外二次抗体, 在室温下的60分钟, 在黑暗中的振动筛上探测膜。在 tbs/T 和 2 x 5 分钟的 tbs 中冲洗 3 x 10 分钟的膜. </li>
		<li> 在适当的激发波长上使用红外成像系统扫描膜 ( 如红外线染料的 700 nm (红色通道) 680 山羊 anti-mouse igg (h + l) 第二和 800 nm (绿色通道) 用于红外染料790驴兔 igg (h + l) 第二级). </li>
		<li> 使用成像软件对信号强度进行量化, 并按照制造商和 #39 的指示执行密度测量, 确保 auto-background 设置为整个背景的平均值. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Taylor, J. P., Hardy, J., Fischbeck, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+Proteins+in+Neurodegenerative+Disease.">Toxic Proteins in Neurodegenerative Disease.</a> Science. 296 (5575), 1991 (1991).</li><li>Ross, C. A., Poirier, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregation+and+neurodegenerative+disease.">Protein aggregation and neurodegenerative disease.</a> Nature Medicine. 10, S10-S17 (2004).</li><li>Ram&#237;rez-Alvarado, M., Merkel, J. S., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+exploration+of+the+influence+of+the+protein+stability+on+amyloid+fibril+formation+in+vitro.">A systematic exploration of the influence of the protein stability on amyloid fibril formation in vitro.</a> Proc. Natl. Acad. Sci. 97 (16), 8979-8984 (2000).</li><li>Hamley, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyloid+Beta+Peptide:+A+Chemist's+Perspective.+Role+in+Alzheimer's+and+Fibrillization.">The Amyloid Beta Peptide: A Chemist's Perspective. Role in Alzheimer's and Fibrillization.</a> Chem Rev. 112 (10), 5147-5192 (2012).</li><li>Diner, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+Properties+of+the+Small+Nuclear+Ribonucleoprotein+U1-70K+in+Alzheimer+Disease.">Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease.</a> J. Biol. Chem. 289 (51), 35296-35313 (2014).</li><li>Gozal, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+Analysis+Reveals+Novel+Components+in+the+Detergent-Insoluble+Subproteome+in+Alzheimer's+Disease.">Proteomics Analysis Reveals Novel Components in the Detergent-Insoluble Subproteome in Alzheimer's Disease.</a> J. Proteome Res. 8 (11), 5069-5079 (2009).</li><li>Hales, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+detergent-insoluble+brain+proteome+linked+to+amyloid+and+tau+in+Alzheimer's+Disease+progression.">Changes in the detergent-insoluble brain proteome linked to amyloid and tau in Alzheimer's Disease progression.</a> PROTEOMICS. , (2016).</li><li>Julien, C., Bretteville, A., Planel, E., Sigurdsson, E. M., Calero, M., Gasset, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Amyloid Proteins: Methods and Protocols. , 473-491 (2012).</li><li>Nizhnikov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Screening+for+Amyloid+Proteins.">Proteomic Screening for Amyloid Proteins.</a> PLoS ONE. 9 (12), e116003 (2014).</li><li>Seyfried, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+detergent-insoluble+brain+proteome+in+frontotemporal+lobar+degeneration+using+SILAC+internal+standards.">Quantitative analysis of the detergent-insoluble brain proteome in frontotemporal lobar degeneration using SILAC internal standards.</a> J. Proteome Res. 11 (5), 2721-2738 (2012).</li><li>Woltjer, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+determination+of+widespread+detergent+insolubility,+including+A&#946;+but+not+tau,+early+in+the+pathogenesis+of+Alzheimer's+disease.">Proteomic determination of widespread detergent insolubility, including A&#946; but not tau, early in the pathogenesis of Alzheimer's disease.</a> FASEB J. , (2005).</li><li>Miake, H., Mizusawa, H., Iwatsubo, T., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+Characterization+of+the+Core+Structure+of+&#945;-Synuclein+Filaments.">Biochemical Characterization of the Core Structure of &#945;-Synuclein Filaments.</a> J. Biol. Chem. 277 (21), 19213-19219 (2002).</li><li>Hasegawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+&#945;-Synuclein+Is+Ubiquitinated+in+&#945;-Synucleinopathy+Lesions.">Phosphorylated &#945;-Synuclein Is Ubiquitinated in &#945;-Synucleinopathy Lesions.</a> J. Biol. Chem. 277 (50), 49071-49076 (2002).</li><li>Neumann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitinated+TDP-43+in+Frontotemporal+Lobar+Degeneration+and+Amyotrophic+Lateral+Sclerosis.">Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis.</a> Science. 314 (5796), 130 (2006).</li><li>Bishof, I., Diner, I., Seyfried, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Intrinsically+Disordered+Low+Complexity+Domain+is+Required+for+U1-70K+Self-association.">An Intrinsically Disordered Low Complexity Domain is Required for U1-70K Self-association.</a> FASEB J. 29 (Suppl 1), (2015).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+stageing+of+Alzheimer-related+changes.">Neuropathological stageing of Alzheimer-related changes.</a> Acta Neuropathol. 82 (4), 239-259 (1991).</li><li>Noguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Patient-derived,+Toxic,+High+Mass+Amyloid+&#946;-Protein+(A&#946;)+Assembly+from+Alzheimer+Disease+Brains.">Isolation and Characterization of Patient-derived, Toxic, High Mass Amyloid &#946;-Protein (A&#946;) Assembly from Alzheimer Disease Brains.</a> J. Biol. Chem. 284 (47), 32895-32905 (2009).</li><li>Bai, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+small+nuclear+ribonucleoprotein+complex+and+RNA+splicing+alterations+in+Alzheimer's+disease.">U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.</a> Proc. Natl. Acad. Sci. 110 (41), 16562-16567 (2013).</li><li>Klunk, W. E., Pettegrew, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+&#946;-Amyloid+Protein+Is+Covalently+Modified+when+Dissolved+in+Formic+Acid.">Alzheimer's &#946;-Amyloid Protein Is Covalently Modified when Dissolved in Formic Acid.</a> J Neurochem. 54 (6), 2050-2056 (1990).</li><li>Yang, L. -. S., Gordon-Krajcer, W., Ksiezak-Reding, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+Released+from+Paired+Helical+Filaments+with+Formic+Acid+or+Guanidine+Is+Susceptible+to+Calpain-Mediated+Proteolysis.">Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis.</a> J Neurochem. 69 (4), 1548-1558 (1997).</li><li>Bai, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+small+nuclear+ribonucleoprotein+complex+and+RNA+splicing+alterations+in+Alzheimer's+disease.">U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 110 (41), 16562-16567 (2013).</li><li>Hales, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+small+nuclear+ribonucleoproteins+(snRNPs)+aggregate+in+Alzheimer's+disease+due+to+autosomal+dominant+genetic+mutations+and+trisomy+21.">U1 small nuclear ribonucleoproteins (snRNPs) aggregate in Alzheimer's disease due to autosomal dominant genetic mutations and trisomy 21.</a> Mol Neurodegener. 9 (1), 15 (2014).</li><li>Xiong, L. -. W., Raymond, L. D., Hayes, S. F., Raymond, G. J., Caughey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+change,+aggregation+and+fibril+formation+induced+by+detergent+treatments+of+cellular+prion+protein.">Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein.</a> J Neurochem. 79 (3), 669-678 (2001).</li><li>Mirra, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Consortium+to+Establish+a+Registry+for+Alzheimer's+Disease+(CERAD).+Part+II.+Standardization+of+the+neuropathologic+assessment+of+Alzheimer's+disease.">The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease.</a> Neurology. 41 (4), 479-486 (1991).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Guo, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+pathological+tau+conformers+from+Alzheimer's+brains+transmit+tau+pathology+in+nontransgenic+mice.">Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice.</a> J. Exp. Med. 213 (12), 2635-2654 (2016).</li><li>Matveev, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+subfraction+of+A&#946;+is+responsible+for+the+high-affinity+Pittsburgh+compound+B-binding+site+in+Alzheimer's+disease+brain.">A distinct subfraction of A&#946; is responsible for the high-affinity Pittsburgh compound B-binding site in Alzheimer's disease brain.</a> J Neurochem. 131 (3), 356-368 (2014).</li></ol>

作者没有什么可透露的。

在此, 我们介绍并讨论了一个简化的单步洗涤剂分馏协议, 适用于广泛的各种实验应用范围从质量谱蛋白质组学分析到功能蛋白折叠测定和西部印迹。5,6,7,10这种方法可能是最有效的, 当用于研究神经退行性 proteinopathies, 如阿尔茨海默氏症, ALS, 亨廷顿氏症, 朊病毒疾病和各种 tauopathies。与原来的五步顺?...

神经退行性疾病, 如阿尔茨海默氏病 (AD)、帕金森氏病 (PD)、亨廷顿氏病 (HD)、肌萎缩侧索硬化症 (ALS), 以及与之密切相关的朊病毒疾病, proteinopathies 的特点是逐渐积累洗涤剂-不溶性蛋白质聚集在大脑中伴随着认知能力的下降。1,2这种共同的病理特征被认为在这些神经退行性疾病的病因和病理生理学中起着中心作用。2这些聚合体通常由聚合淀粉样纤维组成, 它们由错误蛋白的重复单位构成, 其表现为跨β结构。1,2,3,4生化, 淀粉样蛋白聚合体对化学或热变性和增溶具有很强的抗性,3在传统生化技术中对其纯化、分析和研究提出了独特的挑战。2,5,6,7,8,9,10,11不难的是, 不溶性的蛋白质组分一直是许多研究神经退行性疾病的病理生理学的焦点, 涉及错误蛋白的积累。6,12,13,14
生化分馏技术经常被用来丰富从死后脑匀的洗涤剂不溶性分数。6,12,13,14最常见的方法之一是对组织匀进行顺序提取, 并增加严格的缓冲剂和洗涤剂, 然后离心分解可溶性和不溶性组分。通常使用的顺序分馏协议6,14涉及在无洗涤剂低盐 (LS) 缓冲液中的冷冻组织样品的均一性, 然后依次提取含有高盐, 非离子洗涤剂, 高蔗糖, 离子洗涤剂, 最后 chaotropes 像尿素的缓冲器。6,14此类顺序分馏协议的一个明显的缺点是完成它们所需的大量时间和人工承诺。包括均匀化和离心, 一个典型的五步分馏协议可能需要几个小时甚至几天的时间来完成。4,6,7,10,15,16,17,18此外, 由于许多病理蛋白聚集物仍然不溶于所有, 但最苛刻的条件19,20大多数生成的分数都是有限的值。因此, 使用高盐浓度和非离子洗涤剂的不太严格的分馏步骤基本上是多余的。
以往的研究表明, 离子洗涤剂 n-十二烷基肌 (sarkosyl) 是一个很好的候选的简化一步洗涤剂分馏协议。5,6,12,13,14,21,22,23作为一种变性洗涤剂, sarkosyl 是严格的, 足以溶解在大脑中的绝大多数本机折叠的蛋白质, 没有磷错误蛋白聚合组成β-淀粉样 (a),6,11磷酸化 tau (pTau),6焦油 DNA 结合蛋白 43 (TDP-43),14 α-核,12,13病,23或 U1 小核均一 (U1 snRNPs), 如 U1-70K。5,21,22由于 sarkosyl 比无处不在的阴离子洗涤剂十二烷基硫酸钠 (sds) 的严格, 它保留了不太坚固的聚形式的错误蛋白骨料, 无法抵御 sds 治疗。9
以前, 我们描述了一个缩写的洗涤剂分馏协议, 取得的结果可与更费力的顺序分馏方法相媲美。5通过省略较不严格的分馏步骤, 我们得以开发出一种简便的单步分馏协议, 用于丰富死后人脑中的洗涤剂不溶性蛋白聚集物。5本文所述的详细协议非常适合于从西方印迹和大量谱蛋白质组学到功能性蛋白质折叠和聚合播种试验等一系列实验应用。5,6,21

<table><tbody><tr><td>Protease and phosphatase inhibitor cocktail, EDTA-free (100X)</td><td>Thermo Fisher</td><td>78441</td><td>protease &amp;amp; phosphatase inhibitor cocktail</td></tr><tr><td>Sonic Dismembrator System (ultrasonicator)</td><td>Fisher Scientific</td><td>FB505110</td><td>microtip ultrasonicator</td></tr><tr><td>Optimax TLX Ultracentrifuge</td><td>Beckman Coulter</td><td>361545</td><td>refrigerated ultracentrifuge</td></tr><tr><td>TLA120.1 rotor</td><td>Beckman Coulter</td><td>362224</td><td>ultracentrifuge rotor</td></tr><tr><td>500 ul (8 x 34 mm) polycarbonate tubes, thickwall</td><td>Beckman Coulter</td><td>343776</td><td>ultracentrifuge tubes for TLA120.1 rotor</td></tr><tr><td>4X SDS sample buffer</td><td>Home-made</td><td>N/A</td><td>SDS-PAGE</td></tr><tr><td>TCEP solution, neutral pH</td><td>Thermo Fisher</td><td>77720</td><td>reducing agent</td></tr><tr><td>(TBS) blocking buffer</td><td>Thermo Fisher</td><td>37542</td><td>blocking buffer</td></tr><tr><td>(TBS) blocking buffer + 0.05% Tween 20</td><td>Thermo Fisher</td><td>37543</td><td>blocking buffer and antibody diluent</td></tr><tr><td>4-12% Bolt Bis-Tris Plus gels, 10-well</td><td>Thermo Fisher</td><td>NW04120BOX</td><td>precast SDS-PAGE gels</td></tr><tr><td>MES SDS Running Buffer (20X)</td><td>Thermo Fisher</td><td>B0002</td><td>SDS-PAGE running buffer</td></tr><tr><td>N-Lauroylsarcosine sodium salt (sarkosyl)</td><td>Sigma Aldrich</td><td>L5777-50G</td><td>detergent</td></tr><tr><td>Anti-Tau-2 (pan tau) antibody</td><td>Chemicon</td><td>MAB375</td><td>antibodies</td></tr><tr><td>Anti-phospho-threonine 231 Tau antibody</td><td>Millipore</td><td>MAB5450</td><td>antibodies</td></tr><tr><td>Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)</td><td>Thermo Fisher</td><td>MN1020</td><td>antibodies</td></tr><tr><td>Anti-early endosome antigen 1 (EEA1) antibody</td><td>abcam</td><td>ab2900</td><td>antibodies</td></tr><tr><td>Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody</td><td>Thermo Fisher</td><td>A21058</td><td>antibodies</td></tr><tr><td>Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody</td><td>Thermo Fisher</td><td>A11374</td><td>antibodies</td></tr><tr><td>iBlot2 Dry Blotting System</td><td>Thermo Fisher</td><td>IB21001</td><td>Gel transfer</td></tr><tr><td>iBlot2 Transfer Stacks, Nitrocellulose, mini</td><td>Thermo Fisher</td><td>IB23002</td><td>Gel transfer</td></tr></tbody></table>

enrichment of detergent-insoluble protein aggregates from human postmortem brain

在本研究中, 我们描述了一个缩写的单步分馏协议, 以丰富的洗涤剂不溶性蛋白聚集从人死后大脑。离子洗涤剂 n-十二烷基肌 (sarkosyl) 有效地 solubilizes 本机折叠蛋白质在脑组织允许丰富的洗涤剂不溶性蛋白聚集从广泛的神经退行性 proteinopathies, 如阿尔茨海默病 (AD), 帕金森氏症和肌萎缩侧索硬化症, 和朊病毒疾病。人的控制和 AD 死后的脑组织均匀化和沉淀的离心存在的 sarkosyl 丰富的洗涤剂不溶性蛋白骨料, 包括病理磷酸化 tau, 纤维的核心组成部分在广告中缠结。西部印迹显示了聚合磷酸化 tau 和洗涤剂可溶性蛋白, 早期 Endosome 抗原 1 (EEA1) 在控制和 AD 脑的差异溶解度。蛋白质组分析还显示, 与对照组相比, 在 AD 脑 sarkosyl 不溶性的片段中, β淀粉样蛋白 (a)、tau、snRNP70 (U1-70K) 和载脂蛋白 E (脂蛋白) 的富集与以前的组织分馏策略相一致.因此, 这一简单的浓缩协议是理想的广泛的实验应用范围从西方印迹和功能蛋白 co-aggregation 化验, 以大规模谱蛋白质组学。

缩写的单步 sarkosyl 分馏协议是用来丰富从控制和 AD 死后大脑 (图 1) 的洗涤剂不溶性蛋白集料。S1、S2 和 P2 分数的蛋白质通过 SDS-页解决, 固定在马斯亮蓝色定影液中15分钟, 然后用马斯亮亮蓝 G-250 染色缓冲液进行温和染色。悬浮步骤是可选的, 因为 S2 馏分中的蛋白质含量是不可检测的 (见 1.1.14)。西方印迹分析 ( 图 2A</strong...

Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

本文介绍了一种从人死后大脑中浓缩洗涤剂不溶性蛋白集料的简化分馏方法。

人死后脑中洗涤剂不溶性蛋白的富集

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

enrichment-detergent-insoluble-protein-aggregates-from-human

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

12.0K 次观看.  Emory School of Medicine. 本文介绍了一种从人死后大脑中浓缩洗涤剂不溶性蛋白集料的简化分馏方法。

视频: Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot检测人脑皮层组织中的Aβ多聚匀浆使用抗原表位检索

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

科研

神经科学

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

在正常人脑分离的可溶性和不溶性朊蛋白低聚物

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

医学

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

从人死后脑组织神经元细胞核分离

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

生物学

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

用于评估啮齿动物和人类脑组织中行为素聚合状态的基于时间的荧光光谱分析

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer&#39;s Disease (AD)/Frontotemporal Dementia (FTD), and Huntington&#39;s Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

This protocol describes an optimized workflow for nuclei isolation and super-resolution structured illumination microscopy to evaluate individual nucleoporins within the nucleoplasm and NPCs in induced pluripotent stem cell derived neurons and postmortem human tissues.

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

从脑组织提取物中分离淀粉样蛋白原纤维

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

Isolation of Detergent-Insoluble Protein Aggregates From Human Postmortem Brain Tissue

Source: Diner, I., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55835">Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain.</a> J. Vis. Exp. (2017).
This video explains the process of isolating and enriching detergent-insoluble protein aggregates from frozen postmortem human brain tissue. It demonstrates key steps, including homogenization, detergent treatment, sonication, and ultracentrifugation to separate and purify the insoluble aggregates. The final step involves solubilizing the aggregates in urea buffer for further analysis.

从人死后脑组织中分离去污剂不溶性蛋白质聚集体

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;&#160;
1. Homogenization and ...