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 agregasyonu, neden çünkü bu adım gereklidir. Filtrasyon ve SEC gibi diğer yöntemleri de agrega-PICUP 6 için hazırlıklarını elde etmek için kullanılabilir. </li><li> HFIP ile peptitler tedavi için yeterli koruma (HFIP uçucu ve zehirli) giyen bir davlumbaz içinde buz üzerinde HFIP konteyner ön soğutun. 250 ml şişe soğutma genellikle 10-15 dakika gerektirir. Peptid lyophilizates içeren 0,5 mM nominal peptid konsantrasyonu elde etmek için önceden soğutulmuş tüplere HFIP ekleyin. </li><li> Oda sıcaklığında 5 dakika boyunca bir su banyosu sonikatör peptid çözümler sonikasyon. </li><li> Yavaşça vorteksleyin ve tüpler oda sıcaklığında 30 dakika inkübe edin. </li><li> Buz (1 dakika için) tüpler Chill ve 10-50-ul alikotları etiketli 0.6 ml az emici tüplerin içine çözümler bölmek. </li><li> Nazik bir azot akımı altında buharlaşma HFIP çıkarın, ya da gece davlumbaz tüpleri açık bırakın. Gecede buharlaşma için, açık tüpleri rafa yerleştirin ve Kimwipes toz ve partikül kontaminasyonu engellemek için büyük bir levha ile onları karşılamak. </li><li> , Bir lyophilizer vakumlu kalan HFIP, ya da 30 dakika santrifüj konsantratör Exsiccate ya da 2 saat süreyle bir vakum girişine bağlı bir exsiccator Nihai ürün mikrofuge&#39;de tüplerin alt kısmında bir peptid film olacak. Eğer düzgün exsiccated, tüpler -20 veya -80 ° C, uzun süre (ay) hava geçirmeyen saklanabilir. </li></ol> 2. HFIP ile tedavi edilen peptitler çözücü ve fotoğraf çapraz bağlama <ol><li> Çapraz bağlama reaksiyonlar, çapraz bağlama ve soğutma reaktifler hazırlamak için bir ihtiyacı için HFIP ile tedavi edilen peptitler çözülebilmesinde önce. </li><li> Amonyum persülfat tartılır (APS, Sayın 228,2 g / mol) ve 10 mM sodyum fosfat, pH 7.4, 20 mM solüsyon hazırlanır. Çözüm netleşene kadar bir vorteks ile karıştırın. </li><li> 10 mM sodyum fosfat, pH 7.4, 1 mM çözüm Tris (2,2-bipyridyl) dichlororuthenium (II) hekzahidrat (RuBpy, Bay 748,63 g / mol) hazırlayın. Bir vorteks ile karıştırın ve tam dağılması doğrulamak. Tüp alüminyum folyo kullanarak ışık koruyun. </li><li> Çapraz bağlama sonrası SDS-PAGE analizi için uygun bir ısıl işlem reaktif% 5 β-mercaptoethanol 2 × SDS-PAGE numune tamponu. Alternatif olarak, 1 M dithiothreitol (DTT, Bay 154.5 g / mol) deiyonize su veya uygun bir tampon kullanılabilir. </li><li> HFIP ile tedavi edilen peptid filmler seyreltik NaOH ilk çözülür ve daha sonra sodyum fosfat tamponu ~ 30 mcM peptid konsantrasyonu elde etmek için eklenir. Ekle 60 mM NaOH, NaOH ve su sırasıyla 10 ve% 45, son hacim teşkil ettiğini gibi peptid film içeren tüpe deiyonize su ile izledi. Ucu, yukarı ve aşağı pipetleme karıştırın ve bir su banyosunda sonikatör 5 dakika boyunca sonikasyon kullanarak mikrofuge&#39;de tüp iç duvarları kapalı peptid filmi Pençe. </li><li> 45% 20 mM sodyum fosfat, pH 7.4, pipetleme ile karıştırın ve 10 dakika 16.000 g de santrifüj ekleyin. Negatif kontrol için su verme reaktifi ile karışık çizgilerini iptal bağlantılı peptidler hacimde bir kenara koyun. </li><li> 1 s kamera deklanşör gecikmesi ayarlayın. Yüksek ışınlama zamanlarda, geniş radikal reaksiyonlar protein düşmesine neden olabilir. Fotoğraf makinesi obtüratör yükleyin. Işınlama süresi yeni bir protein ile yöntemi kullanılarak optimize edilmesi gerekebilir. </li><li> Tipik bir PICUP reaksiyonu 20 ul reaksiyon hacmi yapılır. Peptid çözüm 18 ul açık, ince duvarlı, 0.2 ml PCR tüpü içine aktarın. </li><li> Peptid çözüm için, 1 1 ul APS takip ul RuBpy ekleyin ve reaktifler pipetleme (reaksiyon karışımı RuBpy ve APS son konsantrasyonu 0.05 ve 1 mM, sırasıyla olacak) karıştırın. </li><li> Reaksiyon tüpü 1.8 ml&#39;lik cam flakon içinde hızlı bir şekilde yerleştirin. Kamera gövdesi önünde bağlı körük içindeki şişe yerleştirin. Lens koruyucusu takın ve örnek körük içinde 1 sn için ışınlanmış, böylece deklanşöre basın. </li><li> Örnek ışınlama sonra, hızla flakon flakon körük ve PCR tüp almak. 1 ul DTT ya da 10 ul azaltarak SAYFA numune tamponu ekleyerek reaksiyon hızla gidermek. Reaksiyon peptid her kısım için tekrarlayın. </li><li> 7 günden daha uzun ya da SDS-PAGE ve gümüş boyama ile analizden önce buz üzerinde tutulmalıdır tepkime saklama için -20 ° C&#39;de dondurulur. </li></ol> 3. SDS-PAGE ve şerit boyamaçapraz bağlı peptid ürünleri <ol><li> Rutin SDS-PAGE ve gümüş boyama çapraz bağlı peptidler görselleştirmek için yapılmaktadır. </li><li> ~ 130 pmol her kulvar için peptid elde etmek için her kulvar için reaksiyon karışımı 5 ul yükleyin. </li><li> Karşılaştırma için benzer miktarlarda çizgilerini iptal bağlantılı peptitler içerir. Ayrıca peptid bantları moleküler ağırlığı görsel yaklaşım için standart bir protein merdiven içerir. </li><li> Standart bir jel elektroforez cihazları kullanırken jel çalıştırın. Biz Invitrogen XCell SureLock Mini-Hücre sistemi kullanımı ve Invitrogen yayın IM-1002, Novex Pre-Cast Jel Elektroforez Kılavuzu göre gümüş boyama gerçekleştirmek. </li></ol> Bölüm 4: Temsilcilik Sonuçları (Şekil 1) <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="Şekil 1" /> Şekil 1: çizgilerini iptal-bagli gösteren gümüş lekeli poliakrilamid jeller (+ -) GRF, kalsitonin, Aβ40 ve Aβ42 ve foto-çapraz bağlı () SDS-PAGE ve PICUP oluşturulan Aβ40 oligomerler gümüş boyama analizleri yüksek oligomerler 7 bolluk içinde keskin bir düşüş takip tetramer yoluyla monomer yaklaşık benzer bant yoğunlukları göstermektedir. Çizgilerini iptal bağlı Aβ40 monomer 7 ile tutarlı bir Bay ile geçirir. Aβ42 ayrı bir oligomer boyut dağılımı gösterir. Aβ42 oligomerler 2-3 8 grupları oluşturmaktadır. Birinci grup, trimer yoluyla monomer, artan oligomerlerdir sipariş ile azalan yoğunluk gösterir. İkinci grupta ise, bir Gauss dağılımı, pentamer ve hexamer maksimum tetramer ve heptamer arasında görülmektedir. Burada gösterilen üçüncü grup, Bay ~ 30,000-60,000 Da 8 oligomerler içerir. Bu yüksek Bay oligomerler genellikle SEC izole LMW Aβ42 kullanarak gözlenen ama peptid filtrasyon veya HFIP tedavisi 6 tarafından hazırlanmıştır. Çizgilerini iptal-bağlantılı Aβ42 ağırlıklı olarak iki grup, bir monomer bant ve 5,9 SDS tarafından uyarılan bir eserdir, geniş bir trimer / tetramer bant, üretir. Kalsitonin dimerleri, trimerler ve tetramers 7 pre-varlığını düşündüren, tetramer yoluyla bölge monomer monoton bir üstel azalma uzaklaşmakta oligomer boyutu dağılımı verir. Kontrolü polipeptid, GRF, dimer oligomerlerinin belirgin göreceli miktarları ile katlanarak artan moleküler kütlesi azaldığı gibi hexamer kadar monoton oligomer dağıtım üretir. Diğer deneylerde, yüksek oligomerler görüntülenmiştir 7 de olabilir. Tam sayısı ve göreceli yoğunlukları oligomer bantları biraz gerçek protein konsantrasyonu, jel yüklenen protein miktarı, ve gümüş boyama protokol geliştirme adım için kullanılan zaman bağlı olarak deneyler arasında değişebilir.

<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. 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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=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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Research

JoVE Journal

Biology

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

21.2K Görüntüleme.  University of California, Los Angeles. 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.

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

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Proteinlerin Elektrogöçer Ayırma

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Biyoloji

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Jeller içinde Proteinler Boyama

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Kompleks Biyolojik örnekler seçerek glikozile Peptitler zenginleştirin lektin HPLC Yöntemi

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Lazer Diseksiyon hücreleri Altküme Kaynak gösterilmeden Uygulanan Drosophila Kanat Disk

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Üretilen Rekombinant Disülfid zengin Venom Proteinler Uygulanan Yüksek Randımanlı Sayısal Anlatım Tarama ve Arıtma<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Peptidler ve proteinler ile rekombinant Elastin gibi Polipeptitlerin ve Fusions olmayan kromatografik saflaştırma<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

MALDI-TOF Kütle Spektrometresi ve Özel Veritabanı kullanımı Benzersiz Mağarası Çevre (Kartchner mağaraları, AZ, ABD) Yerli Bakteriler niteleyin için

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Bir Kolorimetrik Altın Nanopartikül Testi Uygulanan Yapı-anahtarlama aptamers seçilmesi için bir yöntem

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Kütle Spektrometresi Analizi için Peptitler içine Dokular ve Sindirim gelen Ekstrasellüler Matriks Proteinleri zenginleştirilmesi

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...