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

लेखकों मानव मस्तिष्क और सामान्य मस्तिष्क के नमूने उपलब्ध कराने के लिए रीढ़ की हड्डी में तरल पदार्थ संसाधन केंद्र (लॉस एंजिल्स, CA) के लिए आभारी हैं. इस अध्ययन के राष्ट्रीय स्वास्थ्य संस्थान (एनआईएच) R01NS062787, CJD फाउंडेशन, एलायंस BioSecure, के रूप में के रूप में अच्छी तरह से उम्र बढ़ने पर विश्वविद्यालय के सेंटर और स्वास्थ्य McGregor फाउंडेशन के समर्थन और राष्ट्रपति के विवेकाधीन कोष (केस वेस्टर्न रिजर्व विश्वविद्यालय) के साथ समर्थन किया था .

1. मस्तिष्क Homogenate और डिटर्जेंट (S2) घुलनशील और अघुलनशील Fractions (P2) की तैयारी <ol><li> 100 मिलीग्राम जमे हुए मानव मस्तिष्क के ऊतकों लो और 100 μl lysis बफर (10 मिमी Tris, 150 मिमी NaCl, 0.5% P-40 Nonidet (एनपी 40), 0.5% EDTA, 7.4 पीएच). </li><li> यह एक ताररहित मोटर द्वार?...

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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>

दृष्टिकोण के संयोजन यहाँ की सूचना लगातार अघुलनशील पीआरपी सी और सामान्य मानव मस्तिष्क से समुच्चय घुलनशील पीआरपी सी oligomers आइसोलेट्स. एक घंटे के लिए 100.000 XG ultracentrifugation एक क्लासिक तरीका है कि व्यापक रूप स?...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> Phenylmethylsulfonyl फ्लोराइड </td><td> सिग्मा Aldrich </td><td> P7626 </td><td> 2-propanol में 72 मिमी </td></tr><tr><td> Proteinase कश्मीर </td><td> सिग्मा Aldrich </td><td> P2308 </td><td> एच 2 हे में 2 मिलीग्राम / मिलीलीटर </td></tr><tr><td> PNGase एफ </td><td> न्यू इंग्लैंड Biolabs </td><td> MWGF200 </td><td></td></tr><tr><td> LE-80K SW55 रोटर ultracentrifuge </td><td> Beckman कल्टर </td><td> भाग सं 365668, 356860 </td><td></td></tr><tr><td> Superdex 200 एचआर मोती </td><td> जीई हेल्थकेयर </td><td> 17-1088-01 </td><td></td></tr><tr><td> AKTA FPLC प्रणाली </td><td> जीई हेल्थकेयर </td><td> 18-1900-26 </td><td></td></tr><tr><td> आण्विक वजन मार्करों </td><td> सिग्मा Aldrich </td><td> MWGF200 </td><td> विविध </td></tr><tr><td> Dynabeads M-280 चुंबकीय मोतियों </td><td> Invitrogen </td><td> 143-01 </td><td></td></tr><tr><td> 3F4 एंटीबॉडी </td><td> Covance </td><td> हस्ताक्षर ३९,६०० </td><td></td></tr><tr><td> 1E4 एंटीबॉडी </td><td> कोशिका विज्ञान </td><td> M1840 </td><td></td></tr><tr><td> तैयार जेल 15% Tris - एचसीएल पूर्व डाली जैल </td><td> जैव रेड </td><td> 345-0020 </td><td></td></tr></tbody></table>

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.

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

सेलुलर prion प्रोटीन की एक नई प्रजाति (पीआरपी<sup&gt; सी</sup&gt;) हाल ही में यहाँ वर्णित तरीकों का उपयोग कर असंक्रमित मानव दिमाग में पहचान की गई है. इन तरीकों को विभिन्न पीआरपी प्रजातियों को अलग करने के लिए इस्तेमाल किया जा सकता है, जबकि उनमें से कुछ भी हैं मानव दिमाग से अलग अन्य misfolded प्रोटीन समुच्चय में उपयोगी हैं.

घुलनशील और अघुलनशील पीआरपी oligomers के सामान्य मानव मस्तिष्क में अलगाव

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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