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。与原来的五步顺序分馏协议相比, 这一步过程可以在一天内完成, 并提供非常相似的结果。5,6,7,9,14,22
该协议的一个重要方面是使用 sarkosyl 作为分馏洗涤剂。与其他洗涤剂类似海卫 X-100 或十二烷基硫酸钠 (SDS), sarkosyl 似乎很适合这项任务;在严格和磷强度方面, 它强大到足以溶解本机折叠的蛋白质的大部分, 同时保持洗涤剂-不, 结构和功能的疾病相关的蛋白质聚合, 本质上是对 SDS 敏感。5,9此外, 与 SDS 不同, sarkosyl 在低温和高盐条件下仍可溶解。
该协议的另一个关键特点是第一轮离心后的第一个 sarkosyl 不溶 (P1) 小球的悬浮。这一洗涤步骤, 使初始颗粒 (P1) 彻底提取和洗涤的任何残留 cross-contaminating 可溶性或总匀浆蛋白, 提供一个纯净的和明确定义的洗涤剂不溶性蛋白质分数。如果没有可选颗粒 (P1) 悬浮和洗涤步骤, 残留可溶性蛋白可以结转到洗涤剂不溶性分数, 可能混淆随后的结果和实验 (即免疫或蛋白质组分析)。
病理错误蛋白聚合体 (即tau、磷头和 a 种) 的不溶性和高分子量性质, 对传统生化技术的分离和分析提出了独特的挑战。5,6,7,8,9,12,17,21,26与可溶性蛋白不同, 不溶性聚集物如 amyloids (a) 和 hyperphosphorylated 头纤维缠结 (NFTs) 不能用传统方法 (如沉淀或快速) 从死后组织中轻易纯化。蛋白质液相色谱 (FPLC)。3,5,6,7,11,12,23,27因此, 缩写的 sarkosyl 分馏协议是唯一的技术, 允许在较短的时间内从死后的大脑中简便地浓缩和分离洗涤剂不溶性的蛋白质聚合体。5,6,8,13,15,17,21,27
虽然可以采用额外的步骤来分离一个纯的、同质的聚合蛋白样本, 但 sarkosyl 分馏协议允许对洗涤剂不溶性蛋白质聚合物进行简便的浓缩, 而时间承诺相对较少。5,6为了支持这一假说, sarkosyl 不溶蛋白质组的蛋白质组研究证实, 绝大多数已知的病理性聚集倾向的蛋白质分成 sarkosyl-不溶性 fraction-in 都是传统的多步骤6,11,21以及我们的新单步分馏方法。5,7,9,10

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 &#38; 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 (8x34mm) 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>1.5 ml polypropylene Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749521-1500</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>cordless motor for Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749540-0000</td>
			<td>homogenization tool</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和 B) 清楚地表明, 在 AD 脑中, 病理上的高分子量磷酸化 tau 骨料是 sarkosyl 不溶的, 很少 pTau 残留在sarkosyl 可溶性馏分。5,6,7,8,10,22相反, 大多数的 pTau 在控制大脑分成 sarkosyl 可溶性分数。5,6,10相比之下, EEA1 主要分成控制和 AD 大脑中的 S1 分数 (图 2C)。在这里, EEA1 作为一个一般标记的大多数本地的, non-pathologic 的蛋白质, 本质上是 sarkosyl 可溶性的5。值得注意的是, 在可溶性分数 (S1) 中, 与 TH S 分数相比, EEA1 的信号稍少一些, 这表明 EEA1 的小池可能 sarkosyl 不溶, 但低于用这种特定抗体检测的西方印迹的限度。同样可能的是, EEA1 在可溶性分数 (S1) 中的信号强度略有下降是由于 EEA1 对 ultracentrifuge 管的非特异性结合, 也可以解释与预期结果稍有偏差。
为了对缩写的单步分馏协议 (图 3A) 的有效性进行基准测试, 对较传统的多级顺序分馏方法 (图 3B), sarkosyl 的相对水平不溶淀粉样前体蛋白 (APP)、tau (MAPT)、小核核糖 U1-70K (snRNP70) 和载脂蛋白 E (载脂蛋白) 通过无标签质谱 (MS) 的蛋白质组学在混合控制 (Ctl), 轻度认知损害 (MCI) 和 AD案例来自以前的研究7,18 (图 3)。洗涤剂不溶性应用的测量有效地代表 a 肽, 因为所有完全胰应用肽定量地映射到 a 区域全长695残滓应用蛋白质7。这两种方法, 在 MCI 和 AD 病例中, 所有四种病理蛋白的 sarkosyl 不溶水平呈上升趋势, 与对照组比较, 与认知衰退和病理负担的强烈相关性一致。总的来说, 这些蛋白质的丰富, 在洗涤剂不溶性分数 (P2) 是显着一致的使用单步18和多级分馏协议7。
然而, 这两种方法之间有细微的差异, 这可能证明有利或有害, 这取决于个别实验的确切性质和目标。在图 3中总结的比较蛋白质组数据可能会根据特定的实验参数和应用程序来通知使用哪种协议。正如人们所期望的, 在样品浓缩和纯度之间有一个一般的权衡, 简化的分馏协议提供了比多步骤方法更少的蛋白质损失的更丰富的样品。
例如, 使用简化的协议 (图 3A) 编写的控制不溶性分数比通过多步骤方法制备的疾病相关蛋白的背景水平略高。反之, 单步分馏技术可能有利于丰蛋白, 因为整体样本损失明显减少。例如, 通过缩写的协议 (图 3A) 编写的不溶性分数中, MCI 中 sarkosyl 不溶的载脂蛋白的相对富集显著高。
尽管这两种方法都足以观察到与疾病相关或病理错误蛋白聚合体的任何显著增加, 但多步骤方法的大量和广泛的分馏和洗步可能会产生更清洁和更具体的蛋白质组特征。然而, 我们的数据证实单步分馏法与发表的发现一致, 如 tau 纤维缠结 (NFTs), a 斑块和病朊 (PrSc) 的病理骨料不溶于 sarkosyl,而其本身折叠的对应物仍可溶。5,6,7,8,10,11,23,26
通过计算分割成不溶性控制 (n=3) 和 AD (n=3) 脑 (表 2) 的蛋白质的相对数量, 只有 ~ 10% 的总蛋白池是 sarkosyl 不溶的。当5毫克的脑匀浆总蛋白 (TH S) 的分次, 10.4% 和11.3% 的蛋白质组划分到洗涤剂不溶性分数的控制和 AD 的情况下, 分别。虽然 AD 脑中富含的 sarkosyl 不溶蛋白的总量略高于对照, 但两组间没有显著差异 (p = 0.703)。这表明蛋白质折叠和聚集在 AD 脑中并不普遍, 而是局限于一个狭窄和特定的蛋白质子集, 如 tau、a 和 U1 snRNPs。7,21,22

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55835/55835fig1.jpg"> 
图 1: Sarkosyl 分馏方案.(a) 死后人脑在低盐缓冲液中均质化, 然后将 sarkosyl 和氯化钠分别添加到1% 瓦/v 和0.5 米的最终浓度, 并在冰上孵育15分钟。在超声后, 匀离心在 18万x g上分馏, 30 分钟, 提供 sarkosyl 可溶性上清 (S1) 和 sarkosyl 不溶性颗粒 (P1)。P1 颗粒 (可选) 用萨克缓冲区和 re-sedimented 离心, 以支付最终 sarkosyl 不溶性颗粒 (P2)。这 P2 分数是溶解在尿素缓冲30分钟室温, 其次是 3 x 5 s 超声与清洁 mircotip 探头。(B) 蛋白质 (20 µg) 从 TH S, S1, S2 (10 µL) 和 P2 分数, 解决了 SDS 页, 固定15分钟马斯亮蓝色定影液后, 温和染色马斯亮明亮的蓝色 G-250 染色缓冲过夜在室温。<a href="//ecsource.jove. href=""></a href="//ecsource.jove.>com/文件/ftp_upload/55835/55835fig1large. jpg "目标 =" _blank ">> 请点击这里查看这个数字更大的版本。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55835/55835fig2.jpg"> 
图 2: 在 AD 脑的洗涤剂不溶性部分中磷酸化 tau 的富集.(A和B)蛋白 (40 微克) 的总 (TH), 可溶性 (S1) 和不溶性 (P2) 分数被涂抹在苏氨酸 231 (pT231) 或 AT8 (pSer202, pThr205) 的抗体对 tau 磷酸化, 以证明丰富的病理磷 tau 物种在AD 脑的不溶性分数 (P2)。在 AD 脑中, 高分子量聚合头完全分割成洗涤剂不溶 (P2) 分数。(C) EEA1 是一种可溶性蛋白标记物, 是控制和 AD 脑总 (TH S) 和可溶性 (S1) 分数的相对负荷控制。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55835/55835fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55835/55835fig3.jpg"> 
图 3: 使用单步或多级分馏协议的病理 AD 蛋白质的蛋白质组分析.有代表性的 sarkosyl 不溶蛋白应用 (a), tau, snRNP70 (U1-70K) 和载脂蛋白跨不同疾病之间的混合控制, MCI 和 AD 案例使用单步 (A) 或多步骤 (B) sarkosyl分馏法。对于这两种数据集, 蛋白质水平是由肽谱匹配 (psm), 这些确定的蛋白质, 并规范化设置最大值为100。psm 从控制、MCI 和 AD 案例 (n=5 ) 分别用于 single-set 数据集。对于多步方法, 分析了来自两个控制池的 psm、MCI 和 AD 案例 (n=5 )。误差线表示平均值± S.E.M. 的值<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55835/55835fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% sarkosyl 解决方案: 10 克 n-十二烷基肌钠盐每100毫升的 ddH2O (在 RT 隔夜搅拌)</td>
		</tr>
		<tr>
			<td>低盐 (LS) 缓冲: 50 mm HEPES pH 7.0, 250 毫米蔗糖, 1 毫米 EDTA</td>
		</tr>
		<tr>
			<td>Sarkosyl (沙克) 缓冲区:LS 缓冲器 + 1% (w/v) sarkosyl + 0.5 米氯化钠</td>
		</tr>
		<tr>
			<td>尿素缓冲液 (存储在-20 ° c): 50 毫米三盐酸盐 pH 值 8.5, 8M 尿素, 2% SDS</td>
		</tr>
		<tr>
			<td>4X SDS 加载缓冲器: 200 毫米三盐酸盐 ph 6.8, 40% 甘油, 8% 十二烷基 suflate (SDS), 0.4% 溴蓝色 (BPB), 20 mm TCEP ph 7.0 在 ddH2O</td>
		</tr>
		<tr>
			<td>马斯亮固定缓冲器: 40% 甲醇, 10% 乙酸在 ddH2O</td>
		</tr>
		<tr>
			<td>马斯亮明亮的蓝色 G-250 染色缓冲: 25% 甲醇, 5% 乙酸, 0.1% 马斯亮蓝色 G-250 在 ddH2O</td>
		</tr>
		<tr>
			<td>马斯亮 destain 缓冲区: 25% 甲醇, 5% 乙酸在 ddH2O</td>
		</tr>
		<tr>
			<td>1X 三缓冲盐水 (TBS): 50 毫米三盐酸盐 pH 7.45, 150 毫米氯化钠在 ddH2O</td>
		</tr>
		<tr>
			<td>1X 三缓冲盐水与吐温 20 (TBS/T): 50 毫米三盐酸盐 pH 7.45, 150 毫米氯化钠, 0.1% 吐温20在 ddH2O</td>
		</tr>
		<tr>
			<td>1X 阻塞缓冲器 (BB): 50 mm 三盐酸盐 pH 7.45, 150 毫米氯化钠, 1% 专有蛋白质 (见材料列表), 750 mm Methylchloroisothiazolinone 在 ddH2O</td>
		</tr>
		<tr>
			<td>1X 阻塞缓冲器, 0.05% 补间 20 (BB/T): 50 毫米三盐酸盐 pH 7.45, 150 毫米氯化钠, 1% 专有蛋白 (见材料列表), 0.05% 吐温 20, 750 毫米 Methylchloroisothiazolinone 在 ddH2O</td>
		</tr>
	</tbody>
</table>
表 1: 缓冲区列表。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>示例</td>
			<td>卷样 (µL)</td>
			<td>浓度 (µg/µL)</td>
			<td>总蛋白 (µg)</td>
			<td>平均总蛋白 (µg) 和 S.E.M。</td>
			<td>% TH S (5 毫克)</td>
		</tr>
		<tr>
			<td>Ctl 1</td>
			<td>70</td>
			<td>9.89</td>
			<td>692。3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ctl 2</td>
			<td>70</td>
			<td>7.38</td>
			<td>516。6</td>
			<td>518.7 (±99.63)</td>
			<td>10.4%</td>
		</tr>
		<tr>
			<td>Ctl 3</td>
			<td>70</td>
			<td>4.96</td>
			<td>347。2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>广告1</td>
			<td>70</td>
			<td>9.77</td>
			<td>683。9</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>广告2</td>
			<td>70</td>
			<td>6.67</td>
			<td>466。9</td>
			<td>567.0 (±63.20)</td>
			<td>11.3%</td>
		</tr>
		<tr>
			<td>广告3</td>
			<td>70</td>
			<td>7.86</td>
			<td>550。2</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
表 2: sarkosyl 不溶馏分中的蛋白质量 (P2).平均而言, 分割成 sarkosyl 不溶性分数的蛋白质的百分比 (n= 3) 和 AD (n= 3) 的大脑分别为10.4% 和11.3%。从对照和 AD 脑 sarkosyl 不溶蛋白的数量上没有统计学意义上的差异, 由学生的t测试 (p = 0.703) 和平均值 (S.E.M.) 值的标准误差来证明。

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

Nanyang Technological University

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...