Name: photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides
Uploaded: 2009-01-12T00:00:00
Description: Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Bu çalışma, California Halk Sağlığı Bölümü&#39;nden Larry L. Hillblom Alzheimer Derneği Vakfı, IIRG-07-58.334 ve 07-65798 NIH / NIA, 2005/2E AG027818 ve AG030709 hibe ile desteklendi.

1. Peptid hazırlık <ol><li> Etiketli, silikon kaplı, düşük adsorban mikrofuge&#39;de tüpler içine mikro ve transfer kullanarak liyofilize peptid yaklaşık 100-200 mg tartılır. Burada, insan Aβ40, dizileri, Aβ42, kalsitonin ve GRF kullanın. </li><li> Burada, peptidler 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) ile homojen, toplam hazırlıkları elde etmek için ön tedavi kullanın. Önceden oluşmuş agrega deneyleri 5 arasında kötü tekrarlanabilirlik neden amiloidojenik proteinlerin hızlı...

<ol>
	<li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proc. Natl. Acad. Sci. USA. 96, 6020-6024 (1999).</li><li>Kluger, R., Alagic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+cross-linking+and+protein-protein+interactions&#8212;a+review+with+illustrative+protocols.">Chemical cross-linking and protein-protein interactions&#8212;a review with illustrative protocols.</a> Bioorg. Chem. 32, 451-472 (2004).</li><li>Tomohiro, T., Hashimoto, M., Hatanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+chemistry+and+biology:+development+of+multifunctional+photoaffinity+probes.">Cross-linking chemistry and biology: development of multifunctional photoaffinity probes.</a> Chem. Rec. 5, 385-395 (2005).</li><li>Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+protein+oligomers&#8212;what+you+see+is+not+always+what+you+get.">Neurotoxic protein oligomers&#8212;what you see is not always what you get.</a> Amyloid. 12, 88-95 (2005).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+aggregate-free,+low+molecular+weight+amyloid-&#946;+for+assembly+and+toxicity+assays.">Preparation of aggregate-free, low molecular weight amyloid-&#946; for assembly and toxicity assays.</a> Methods Mol. Biol. 299, 3-9 (2005).</li><li>Bitan, G., Lomakin, A., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. D., Breese, R., Dodson, E. C., Acton, P., Keller, P. M., Yeager, M., Wang, H., Shughrue, P., Kinney, G., Joyce, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+state+characterization+of+amyloid+&#946;-derived+diffusible+ligands.">Solution state characterization of amyloid &#946;-derived diffusible ligands.</a> Biochemistry (Mosc). 45, 15157-15167 (2006).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+photochemical+cross-linking&#8212;a+new+tool+for+studies+of+metastable,+amyloidogenic+protein+assemblies).">Rapid photochemical cross-linking&#8212;a new tool for studies of metastable, amyloidogenic protein assemblies).</a> Acc. Chem. Res. 37, 357-364 (2004).</li><li>Piening, N., Weber, P., Hogen, T., Beekes, M., Kretzschmar, H., Giese, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+crosslinking+of+prion+protein+oligomers+and+prions.">Photo-induced crosslinking of prion protein oligomers and prions.</a> Amyloid. 13, 67-77 (2006).</li><li>Li, H. T., Lin, X. J., Xie, Y. Y., Hu, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+events+of+&#945;-synuclein+oligomerization+revealed+by+photo-induced+cross-linking.">The early events of &#945;-synuclein oligomerization revealed by photo-induced cross-linking.</a> Protein Pept. Lett. 13, 385-390 (2006).</li><li>Vollers, S. S., Teplow, D. B., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Peptide+oligomerization+state+using+rapid+photochemical+crosslinking.">Determination of Peptide oligomerization state using rapid photochemical crosslinking.</a> Methods Mol. Biol. 299, 11-18 (2005).</li></ol>

PICUP, istikrarlı bir protein kompleksleri 2 çalışma ilk geliştirilmiştir . Aβ 10, prion hastalığı ile ilişkili PrP Sc 11 ve α-sinüklein 12 metastabil amiloid protein meclisleri, kantitatif çalışma yöntemi uygulandı. PICUP Deney tasarlama dikkate alınması gereken en önemli faktörler, reaktif stokiyometri, ışınlama süresi ve numune hazırlama prosedürü. Ikinci sorun, büyük ölçüde deneysel verilerin yorumlanması etkiler ise eski iki konu, deneysel optimizasyon gerek...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

Toksik oligomerler amiloidojenik proteinlerin montaj protein misfolding hastalıkları, Alzheimer, Parkinson ve Huntington hastalıkları, kalıtsal Amyotrofik lateral skleroz, ve tip 2 diyabet patogenezinde yeni ufuklar açan bir olaydır. Bu protein meclislerinin metastabil yapısı sayesinde, elektroforez, kromatografi, floresan veya dinamik ışık saçılımı gibi klasik yöntemlerle kantitatif oligomer boyutu dağılımını değerlendirmek için zordur. Amiloidojenik proteinlerin Oligomerleri oligomerler monomerlerin ilgimiz büyük meclisleri içine aynı anda ilişkilendirmek metastabil hangi karışımlar var. PICUP çapraz önce varolan oligomer boyutu dağılımları kovalent çapraz bağlama ve sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) ya da boyut dışlama kromatografisi (SEC) gibi fraksiyon yöntemleri ile kombine edildiğinde, PICUP sağlar anlık görüntülerini oligomer popülasyonları stabilize bağlama. Bu nedenle, PICUP metastabil protein popülasyonlarının görselleştirme ve kantitatif analiz sağlar ve montaj ve deşifre dizisi değişiklikler ve oligomerization arasındaki ilişkileri izlemek için kullanılabilir<sup&gt; 1</sup&gt;. Mekanistik, PICUP Ru foto-oksidasyon içerir<sup&gt; 2 +</sup&gt; Tris (bipyridyl) Ru (II) kompleksi (RuBpy) Ru için<sup&gt; 3 +</sup&gt; Bir elektron alıcısı varlığı görünür ışık ile ışınlama. Ru<sup&gt; 3 +</sup&gt; Bir protein radikal üreten, komşu bir protein molekülünün bir elektron soyutlama yeteneğine sahip güçlü bir elektron oksitleyici<sup&gt; 1,2</sup&gt;. Radikaller, kararsız, son derece reaktif türler ve bu nedenle içi ve moleküller arası reaksiyonlar çeşitli hızla kaybolur. Radikal, daha sonra bir hidrojen atomu kaybetmesi ve istikrarlı, kovalent bağlantılı dimer oluşturur dimerik radikal başka protein oluşturan monomer ile reaksiyona paylaşılmamış elektronu yüksek enerji kullanabilir. Dimer sonra, monomerlerin veya diğer dimerleri üst düzey oligomerler formu ile benzer bir mekanizma aracılığıyla daha fazla tepki gösterebilir. PICUP göreli avantajları diğer fotoğraf ya da kimyasal çapraz bağlama yöntemleri<sup&gt; 3,4</sup&gt; (≤ 1 saniye) kısa tahribatsız görünür ışığa maruz kalma, gerek<i&gt; Ön facto</i&gt; Yerli dizi modifikasyon, sıfır uzunlukta kovalent çapraz bağlama. Buna ek olarak, PICUP sağlar fizyolojik parametreleri de dahil olmak üzere geniş bir pH ve sıcaklık aralıkları içinde proteinlerin çapraz bağlama. Burada üç amiloidojenik proteinler, 40 çapraz-bağlantı PICUP uygulama göstermektedir - ve 42-kalıntı amiloid β-protein türevleri (Aβ40 ve Aβ42), kalsitonin ve kontrol protein, büyüme hormonu salgılatıcı faktör (GRF).

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Foto-bağlı çapraz bağlama değiştirilmemiş proteinleri (PICUP) metastabil protein karışımları oligomer boyutu dağılımı karakterizasyonu sağlar. Biz üç temsilci amiloidojenik peptidler 40 PICUP uygulama göstermektedir - ve 42-amiloid β-protein ve kalsitonin kalıntı formları ve kontrol peptid büyüme hormonu salgılatıcı faktör.

Fotoğraf Bağlı Çapraz Bağlanma Değiştirilmemiş Proteinler (PICUP) amiloidojenik Peptitler Uygulanan

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

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Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

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