Name: isolation of soluble and insoluble prp oligomers in the normal human brain
Uploaded: 2012-10-03T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

“ 作者感谢提供正常的脑组织样本的人的大脑和脊髓液资源中心（洛杉矶，CA）。这项研究是支持由美国国家卫生研究院（NIH）R01NS062787，的克雅氏病基金会，联盟生物安全，以及在大学中心老化和健康的支持麦格雷戈基金会和总统的全权委托基金（Case Western Reserve大学） 。

1。脑匀浆的制备和洗涤剂可溶性（S2）和不溶性的（P2）的馏分<ol><li>取100毫克冷冻人脑组织中，并加入100μl裂解缓冲液中（10mM Tris，150mM的NaCl，0.5％NONIDET P-40（NP-40），0.5％EDTA，pH7.4）中。 </li><li>均质在冰上，用杵由无绳马达驱动，在干冰中冷冻它为5分钟，并再次均质它使用杵先用手驱动，然后由马达，并加入300μl或800微升的裂解缓冲液（这是20 ％或10％总脑匀浆，分别）。 </li><li>以1,000 xg离...

<ol>
	<li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions.+Proc.">Prions. Proc.</a> Natl. Acad. Sci. U.S.A. 95, 13363-13383 (1998).</li><li>Oesch, B., Westaway, D., W&#228;lchli, M., McKinley, M. P., Kent, S. B. H., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., Hood, L. E., Prusiner, S. B., Weissmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cellular+gene+encodes+scrapie+PrP+27-30+protein.">A cellular gene encodes scrapie PrP 27-30 protein.</a> Cell. 40, 735-746 (1985).</li><li>Bolton, D. C., McKinley, M. P., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+that+purifies+with+the+scrapie+prion.">Identification of a protein that purifies with the scrapie prion.</a> Science. 218, 1309-1311 (1982).</li><li>Prusiner, S. B., McKinley, M. P., Bowman, K. A., Bolton, D. C., Bendheim, P. E., Groth, D. F., Glenner, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie+prions+aggregate+to+form+amyloid-like+birefringent+rods.">Scrapie prions aggregate to form amyloid-like birefringent rods.</a> Cell. 35, 349-358 (1983).</li><li>Hope, J., Morton, L. J., Farquhar, C. F., Multhaup, G., Beyreuther, K., Kimberlin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+polypeptide+of+scrapie-associated+fibrils+(SAF)+has+the+same+size,+charge+distribution+and+N-terminal+protein+sequence+as+predicted+for+the+normal+brain+protein+(PrP).">The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP).</a> EMBO J. 5, 2591-2597 (1986).</li><li>Prusiner, S. B., Groth, D. F., Bolton, D. C., Kent, S. B., Hood, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+structural+studies+of+a+major+scrapie+prion+protein.">Purification and structural studies of a major scrapie prion protein.</a> Cell. 38, 127-134 (1984).</li><li>Hall, D., Edskes, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silent+prions+lying+in+wait:+a+two-hit+model+of+prion/amyloid+formation+and+infection.">Silent prions lying in wait: a two-hit model of prion/amyloid formation and infection.</a> J. Mol. Biol. 336, 775-786 (2004).</li><li>Yuan, J., Xiao, X., McGeehan, J., Dong, Z., Cali, I., Fujioka, H., Kong, Q., Kneale, G., Gambetti, P., Zou, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insoluble+aggregates+and+protease-resistant+conformers+of+prion+protein+in+uninfected+human+brains.">Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains.</a> J. Biol. Chem. 281, 34848-34858 (2006).</li><li>Yuan, J., Dong, Z., Guo, J. P., McGeehan, J., Xiao, X., Wang, J., Cali, I., McGeer, P. L., Cashman, N. R., Bessen, R., Surewicz, W. K., Kneale, G., Petersen, R. B., Gambetti, P., Zou, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessibility+of+a+critical+prion+protein+region+involved+in+strain+recognition+and+its+implications+for+the+early+detection+of+prions.">Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions.</a> Cell. Mol. Life Sci. 65, 631-643 (2008).</li><li>Zou, W. Q., Zheng, J., Gray, D. M., Gambetti, P., Chen, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+to+DNA+detects+scrapie+but+not+normal+prion+protein.">Antibody to DNA detects scrapie but not normal prion protein.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 1380-1385 (2004).</li><li>Gambetti, P., Dong, Z., Yuan, J., Xiao, X., Zheng, M., Alshekhlee, A., Castellani, R., Cohen, M., Barria, M. A., Gonzalez-Romero, D., Belay, E. D., Schonberger, L. B., Marder, K., Harris, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+human+disease+with+abnormal+prion+protein+sensitive+to+protease.">A novel human disease with abnormal prion protein sensitive to protease.</a> Ann. Neurol. 63, 697-708 (2008).</li><li>Zou, W. Q., Puoti, G., Xiao, X., Yuan, J., Qing, L., Cali, I., Shimoji, M., Langeveld, J. P., Castellani, R., Notari, S., Crain, B., Schmidt, R. E., Geschwind, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variably+protease-sensitive+prionopathy:+A+new+sporadic+disease+of+the+prion+protein.">Variably protease-sensitive prionopathy: A new sporadic disease of the prion protein.</a> Ann. Neurol. 68, 162-172 (2010).</li><li>Zou, W. Q., Xiao, X., Yuan, J., Puoti, G., Fujioka, H., Wang, X., Richardson, S., Zhou, X., Zou, R., Li, S., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta42+interacts+mainly+with+insoluble+prion+protein+in+the+Alzheimer+brain.">Amyloid-beta42 interacts mainly with insoluble prion protein in the Alzheimer brain.</a> J. Biol. Chem. 286, 15095-15105 (2011).</li><li>Zou, W. Q., Colucci, M., Gambetti, P., Chen, S. G., Potter, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+prion+proteins.">Characterization of prion proteins.</a> Methods in Molecular Biology-Neurogenetics: Methods and Protocols. , 305-314 (2002).</li><li>Tzaban, S., Friedlander, G., Schonberger, O., Horonchik, L., Yedidia, Y., Shaked, G., Gabizon, R., Taraboulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-sensitive+scrapie+prion+protein+in+aggregates+of+heterogeneous+sizes.">Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes.</a> Biochemistry. 41, 12868-12875 (2002).</li></ol>

报告相结合的办法，隔离始终不溶性的朊蛋白的Ç聚集和可溶性PrP的Ç从正常的人脑的低聚物。超速离心法是一个典型的方法，该方法已被广泛用于从可溶的PrP C 14的分离的不溶性的PrP Sc的一小时以100,000 xg离心。虽然它是有效的，其中之一的注意事项是拉离心分离后的上清液（S2）过程中，以避免污染。 IPRPÇ是不同的朊蛋白凝胶调，这是不可能的朊蛋白检?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>苯甲基磺酰氟</td><td> Sigma-Aldrich公司</td><td> P7626 </td><td> 72 mM的2 - 丙醇中</td></tr><tr><td>蛋白酶K </td><td> Sigma-Aldrich公司</td><td> P2308 </td><td>在H 2 O 2毫克/毫升</td></tr><tr><td> PNG酶F </td><td> New England Biolabs公司</td><td> MWGF200 </td><td></td></tr><tr><td>超速离心机LE-80K，SW55转子</td><td> Beckman Coulter公司</td><td>料号：365668，356860 </td><td></td></tr><tr><td> Superdex 200 HR珠</td><td> GE医疗集团</td><td> 17-1088-01 </td><td></td></tr><tr><td> AKTA FPLC系统</td><td> GE医疗集团</td><td> 18-1900-26 </td><td></td></tr><tr><td>分子量标记</td><td> Sigma-Aldrich公司</td><td> MWGF200 </td><td>多变</td></tr><tr><td>的Dynabeads M-280磁珠</td><td> Invitrogen公司</td><td> 143-01 </td><td></td></tr><tr><td> 3F4抗体</td><td>科文斯</td><td> SIG-39600 </td><td></td></tr><tr><td> 1E4抗体</td><td>细胞科学</td><td> M1840 </td><td></td></tr><tr><td>就绪凝胶15％的Tris-HCl预制胶</td><td> Bio-Rad公司</td><td> 345-0020 </td><td></td></tr></tbody></table>

isolation of soluble and insoluble prp oligomers in the normal human brain

朊病毒疾病的发病机制中的中心事件涉及的主机编码朊蛋白PrP的Ç转换成其致病亚型的PrP Sc的1。朊蛋白C是水溶性清洁剂和敏感的蛋白酶K（PK）消化的PrP Sc的 ，而形成不溶于洗涤剂的聚集和部分耐PK 2-6。朊病毒Ç的PrP Sc的转换是已知涉及的构象的蛋白的β-折叠片结构的α-螺旋结构的过渡。然而， 在体内的途径仍然是知之甚少。暂定内源性的PrP Sc的 ，中间的PrP *或“沉默的朊病 ​​毒”，在未受感染的大脑还没有确定。 使用相结合的生物物理和生物化学的方法，我们发现不溶性的朊蛋白的Ç聚集（IPRP C）未感染的哺乳动物的大脑和培养的神经元单元8，9。在这里，我们将详细介绍这些方法的程序，其中包括超速离心法在洗涤缓冲液，蔗糖步骤梯度沉降，尺寸排阻色谱法，IPRP富集基因5蛋白（g5p）特异性结合的结构改变的PrP表格10，和PK-处理。这些方法的组合隔离：不仅不溶性的PrP Sc和朊病毒Ç聚集体，但也可溶性蛋白Ç从正常人类大脑的低聚物。由于这里描述的协议已被用于从受感染的大脑和未感染的大脑IPRP C的隔离双方的PrP Sc的 ，他们为我们提供了一个机会，比较两种异构体之间的物理化学特性，神经毒性，传染性。这样的研究将大大提高我们了解蛋白质病原体的感染。生理学和病理生理学IPRPÇ目前还不清楚。值得注意的是，在一个新的-id的可变entified人类朊病毒疾病被称为蛋白酶敏感的prionopathy，我们发现了一个新的PrP Sc的共享的免疫反应行为IPRP C 11，12和碎片。此外，我们最近表明，IPRP C是主要品种，与β-淀粉样蛋白在阿尔茨海默病13互动。在相同的研究中，这些方法被用来隔离Abeta聚合物和低聚物，在阿尔茨海默氏病13，提示它们的其他神经退行性疾病中涉及的非朊病毒蛋白聚集体中的应用。

Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

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

朊蛋白（PrP的一新种<sup&gt; C</sup&gt;）最近被鉴定使用这里描述的方法在未感染的人脑。这些方法可以用来隔离不同的朊蛋白物质，而其中一些也可在其他错误折叠的蛋白质聚集体从人类的大脑隔离开来。

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 ...

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