Laboratuarda çalışma NIH, ABD Tarım Bakanlığı Gıda ve Tarım Ulusal Enstitüsü, NSF, BARD, Enerji Bakanlığı ve BSF VC hibe tarafından desteklenmektedir

1. İfade ve proteinlerin ekstraksiyonu <ol><li> Farklı şekillerde kendi tespiti için test edilmesi proteinler etiketi. Membran (ProIM), daha büyük boyutta (örneğin, GST) bir etiket ile immobilize ve halkaya yapışmamış etiketi hareketsiz bir negatif kontrol (ProIMnc) olarak kullanılabilir protein etiketleyin. Ya büyük ya da küçük bir etiket ile çözünebilir bir prob (ProSOL) olarak kullanılmak üzere protein etiketleyin. Aynı etiket uygun bir negatif kontrol çözünür protein (ProSOLnc) Sigorta ve tespit edilen bağlama özgüllüğü onaylamak için etkileşim tahlil dahil. </li><li> Etiketli proteinleri ifade etmek protein ekspresyon sistemi, yani E. seçin coli, bakülovirüs vb proteinler potansiyel mesaj translasyonel modifikasyonlar ve verim için ihtiyaç bağlı olarak. </li><li> SDS-PAGE proteinaz inhibitörü kokteyl içeren yükleme tamponu (% 20 gliserol,% 4 SDS, 20 mM Tris-HCl, pH6.8) kullanarak seçim organizma (adım 1.2) ProIM ve ProIM nc ayıklayın. Hücre süspansiyonu bırakın, 15 dakika süreyle oda sıcaklığında 2 merkaptoetanol ve 5 dakika boyunca kaynatın% 0.5 &#39;i ekleyin. </li><li> ProSOL ve ProSOL nc, standart protokolleri 16 kullanarak seçim organizma (adım 1.2) ayıklayın . Bu proteinler adım 4.1 (140 mM NaCl, 10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM DTT,% 1 BSA,% 0.1 Tween 20) bağlayıcı tampon kolayca çözünür olmalıdır. </li><li> Bio-Rad protein tahlil kiti standart bir yöntem kullanılarak rekombinant proteinlerin konsantrasyonu belirleyin. Bio-Rad protein tahlil kiti gibi. Proteinin saflaştırılmış yerine ham özü, tahlil için kullanılması durumunda, Coomassie Brillant Blue R-250 ile ilgili bant densitometrisi SDS-poliakrilamid jel boyanmış ve bilinen kullanarak bu özü ilgi protein konsantrasyonu tahmin referans olarak BSA konsantrasyonları. </li></ol> 2. Membran ProIM ve ProIMnc immobilizasyonu <ol><li> Özler, standart bir protokol 16 göre, SDS-poliakrilamid jel üzerinde ProIM ve ProIM nc 1 mcg içeren her iki takım giderin . </li><li> Elektroforez sonra, 100 ml (60 mM Glisin, 10 mM Tris,% 0.0006 SDS,% 20 MeOH) transfer tampon transferi jel yerleştirin ve yavaşça sallanarak oda sıcaklığında 20 dakika inkübe. </li><li> Nitroselüloz membrana göre standart bir protokol 16 jel bulunan proteinler Electrotransfer. </li></ol> 3. Membrana bağlı proteinler Re-katlama <ol><li> Electrotransfer sonra, 15 dakika için 15 ml tampon membran inkübe A (30 mM Tris-HCl pH7.4,% 0.05 Tween 20) artık SDS kaldırmak için yavaşça sallanarak. </li><li> Dikkatli bir şekilde boşaltılmasının ardından, denatürasyon tampon (7 M guanidin hidroklorür, 2 mM EDTA, 50 mM DTT, 50 mM Tris-HCl, pH 8.3) 25 ml membran aktarın. ve yavaşça sallanarak 2 saat oda sıcaklığında inkübe edin. (Not: denatürasyon tampon inkübe nitroselüloz membran bulanıklaşır). ve yavaşça sallanarak 2 saat oda sıcaklığında inkübe edin. </li><li> 25 TBS ml (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) ve 5 dakika boyunca hafif ajitasyon ile inkübe membran Devret (Not: Bu adım sırasında, orijinal beyaz renkli membran kavuşur). </li><li> Membran bağlayıcı tampon 25 ml (1.2 adım) transferi ve 4 ° C yavaşça sallanarak gece boyunca inkübe edin. </li></ol> 4. ProSOL tarafından ProIM problama <ol><li> Iki şeritler, hem de içeren ProIM ve ProIM nc membran kesin. </li><li> 10 ml taze bağlayıcı tampon ProSOL veya ProSOLnc 1-10 mikrogram ile hazırlık seyreltilmesi ProSOL ve ProSOLnc hibridizasyon çözümleri olun. ProSOL veya ProSOLnc hibridizasyon çözümü her membran transferi ve yavaşça sallanarak oda sıcaklığında 1,5 saat boyunca inkübe edin. </li><li> Hibridizasyon çözümler membranlar çıkarın ve 15 dakika TBS içinde her biri için üç kez yıkayın. </li></ol> 5. Immün protein-protein etkileşim görselleştirme <ol><li> Oda sıcaklığında 1 saat süreyle TBST% 2,5 yağsız süt (10 mM Tris-HCl, 140 mM NaCl,% 0.05 Tween 20, pH 7.4) ile membran engelleyin. TBST% 0,5 yağsız süt üretici tarafından önerilen konsantrasyonda birincil antikor (anti-strepII poliklonal tavşan antikoru) sulandırınız. </li><li> Antikor çözüm bloke membranlar yerleştirin ve oda sıcaklığında 1 saat veya gece boyunca inkübe 4 ° C yavaşça sallanarak. </li><li> 15 dakika için 20 ml TBST membranlar durulayın ve iki kez yavaşça sallanarak oda sıcaklığında 5 dakika. </li><li> TBST% 0,5 yağsız süt üretici tarafından önerilen konsantrasyonda at turp peroksidaz (HRP) ile konjuge sekonder antikoru (anti-tavşan IgG) sulandırınız. Membranlar ikincil antikor yerleştirinçözüm ve yavaşça sallanarak oda sıcaklığında 1 saat süreyle inkübe edin. </li><li> 15 dakika için 20 ml TBST membranlar durulayın ve iki kez yavaşça sallanarak oda sıcaklığında 5 dakika. TBS son durulama sonra, HRP kemilüminesans substrat (örneğin, Millipore Immobilon batı kemilüminesan HRP substrat) kullanarak protein-protein etkileşimleri görselleştirmek. </li><li> 5.4 adımları 5.1 &#39;de açıklanan uygun ikincil antikor takip ProIM, erimiş etiketi için birincil antikor ile aynı membranlar Prob. Görselleştirin ProIM ve ProIMnc protein membran kaplama assay (adım 5.5) elde edilen bantların kimliğini doğrulamak için 5.5 adımı nitelendirdi. Çoğu durumda, bu adımı ile elde edilen sinyal adım 5.5 &#39;ten elde edilen kalıntı sinyali daha güçlü çünkü sıyırma, gerekli değildir. </li></ol> 6. Temsilcisi Sonuçlar: ANK-MP etkileşim tütün epidermal hücreler (Şekil 1A) BiFC gözlendi. MP, bakteri veya bitkilerde ifade yüksek çözünmeyen bir protein olduğu için, protein membran kaplama testi, in vitro (Şekil 1B) bu etkileşimin doğrulamak için kabul edildi. GST-MP (ProIM) veya halkaya yapışmamış GST (ProIM nc) 1 mg protein özleri içeren bir nitroselüloz membran electrotransfer takip SDS-poliakrilamid jel elektroforezi, çözüldü. Bu ProIMs çözünür ANK-strepII (ProSOL), GST-MP, ancak halkaya yapışmamış değil GST probed bağlayıcı sergilenmektedir (Şekil 1B, hat 1, 2, 5, 6 şeritli karşılaştırın). Ayrıca, aynı ProIMs ANK-MP etkileşim özgüllüğü (Şekil 1B, daha gösteren bir ilgisi olmayan ProSOLnc, yani strepII (NADH3-strepII) etiketli Arabidopsis sitoplazmik NADH kinaz, hiçbir bağlayıcı gözlenmemiştir ile probed şerit 3, 4). <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="Şekil 1"/> Şekil 1, in vivo ve in vitro TMV MP tütün ANK Özgül bağlayıcıdır . (A), BiFC tarafından tespit edilen tütün epidermal hücrelerin yaşam ANK-MP etkileşim. MP ve ANK, C-terminal ve sırasıyla YFP N-terminal yarısı erimiş Güçlü YFP sinyali kodlayan genlerin microbombardment tütün epidermiste coexpressed, yeniden inşa edildi. Bu BiFC sinyal plasmodesmata 15 tanı hücre çevre, puncta birikmiş. (B) in vitro ANK-MP etkileşim gibi protein membran kaplama yöntemi ile tespit edildi. GST-MP (ProIM) veya halkaya yapışmamış GST (ProIMnc) 1 mg protein özleri içeren bir nitroselüloz membran üzerine electrotransfer% 15 SDS-poliakrilamid jel, çözüldü. GST-MPProIM ve GSTProIMnc ANK-strepII (ProSOL) 0.5 mg / ml ile inkübe edildi ve ANK bağlama konjuge anti-tavşan IgG + M sekonder antikoru, anti-strepII tavşan poliklonal antikor ile membran problama tespit edildi HRP (1 ve 2 şeritli). GST-MPProIM ne GSTProIMnc ne strepII etiketi (ProSOLnc, şerit 3 ve 4) erimiş alakasız bir protein, Arabidopsis sitoplazmik NADH kinaz ile etkileşim. Bu testte gözlenen grubun kimliğini anti-GST antikor (şerit 5 ve 6) ile membran problama tarafından teyit edildi. ANK-strepII GST-MP ve GST contining protein özleri bulunan kimliği belirsiz proteinler ile reaksiyona membran tampon A ile yıkanır olmadan denatürasyon tamponu ile tedavi edildi, GST-MP ANK bağlama önemini gösteren, kaybolur 3.1 adım önce denatürasyon süreci (şerit 7 ve 8).

<ol>
	<li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9, 789-798 (2002).</li><li>Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+interacting+proteins+by+bimolecular+fluorescence+complementation+in+planta.">Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.</a> J Mol Biol. 362, 1120-1131 (2006).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+Caenorhabditis+elegans+using+bimolecular+fluorescence+complementation+analysis.">Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.</a> Nat Protoc. 3, 588-596 (2008).</li><li>Citovsky, V., Gafni, Y., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+protein-protein+interactions+by+bimolecular+fluorescence+complementation+in+planta.">Localizing protein-protein interactions by bimolecular fluorescence complementation in planta.</a> Methods. 45, 196-206 (2008).</li><li>Ohad, N., Shichrur, K., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+protein-protein+interactions+in+plants+by+bimolecular+fluorescence+complementation.">The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.</a> Plant Physiol. 145, 1090-1099 (2007).</li><li>Zamyatnin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+integral+membrane+protein+topology+in+living+cells.">Assessment of the integral membrane protein topology in living cells.</a> Plant J. 46, 145-154 (2006).</li><li>Citovsky, V., Knorr, D., Schuster, G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P30+movement+protein+of+tobacco+mosaic+virus+is+a+single-strand+nucleic+acid+binding+protein.">The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein.</a> Cell. 60, 637-647 (1990).</li><li>Heinlein, M., Epel, B. L., Padgett, H. S., Beachy, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+tobamovirus+movement+proteins+with+the+plant+cytoskeleton.">Interaction of tobamovirus movement proteins with the plant cytoskeleton.</a> Science. 270, 1983-1985 (1995).</li><li>Tomenius, K., Clapham, D., Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+by+immunogold+cytochemistry+of+the+virus+coded+30+K+protein+in+plasmodesmata+of+leaves+infected+with+tobacco+mosaic+virus.">Localization by immunogold cytochemistry of the virus coded 30 K protein in plasmodesmata of leaves infected with tobacco mosaic virus.</a> Virology. 160, 363-371 (1987).</li><li>Ding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+plasmodesmata+are+specific+sites+of+localization+of+the+tobacco+mosaic+virus+movement+protein+in+transgenic+tobacco+plants.">Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.</a> Plant Cell. 4, 915-928 (1992).</li><li>Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+30+kd+protein+of+tobacco+mosaic+virus:+involvement+in+cell-to-cell+movement+and+dispensability+for+replication.">Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication.</a> EMBO J. 6, 2557-2563 (1987).</li><li>Wolf, S., Deom, C. M., Beachy, R. N., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+protein+of+tobacco+mosaic+virus+modifies+plasmodesmatal+size+exclusion+limit.">Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.</a> Science. 246, 377-379 (1989).</li><li>Waigmann, E., Lucas, W. J., Citovsky, V., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+functional+assay+for+tobacco+mosaic+virus+cell-to-cell+movement+protein+and+identification+of+a+domain+involved+in+increasing+plasmodesmal+permeability.">Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.</a> Proc. Natl. Acad. Sci. USA. 91, 1433-1437 (1994).</li><li>Ueki, S., Spektor, R., Natale, D. M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANK,+a+host+cytoplasmic+receptor+for+the+Tobacco+mosaic+virus+cell-to-cell+movement+protein,+facilitates+intercellular+transport+through+plasmodesmata.">ANK, a host cytoplasmic receptor for the Tobacco mosaic virus cell-to-cell movement protein, facilitates intercellular transport through plasmodesmata.</a> PLoS Pathog. 6, e1001201-e1001201 (2010).</li><li>Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., Wingfield, P. T., Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2011).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP1,+an+Arabidopsis+protein+that+interacts+with+Agrobacterium+VirE2,+is+involved+in+VirE2+nuclear+import+and+Agrobacterium+infectivity.">VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.</a> EMBO J. 20, 3596-3607 (2001).</li><li>Chen, M. H., Sheng, J., Hind, G., Handa, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+the+tobacco+mosaic+virus+movement+protein+and+host+cell+pectin+methylesterases+is+required+for+viral+cell-to-cell+movement.">Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.</a> EMBO J. 19, 913-920 (2000).</li></ol>

Çıkar çatışması ilan etti.

Bu yaklaşım, proteinler en az bağlayıcı tampon kolayca çözünür olduğu ve diğer proteinlerin kombinasyonu 17,18 başarıyla uygulandı proteinler kombinasyonları arasında protein-protein etkileşimleri, test etmek için uygundur . Bu koşullar altında hem de çözünmez proteinler arasındaki iInteractions Bu protokol tarafından test edilemez. Ayrıca, başarılı refolding ProIM testi için kritik öneme sahiptir. Kalan SDS denatürasyon / Yeniden doğallaştırman?...

<table border="1"><tbody><tr><td> Reaktifin Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Protein tahlil kiti </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Proteinaz inhibitörü kokteyl </td><td> SIGMA </td><td> S8820 </td></tr><tr><td> Mini-Protean sistemi </td><td> Bio-RAD </td><td> 165-8000 </td></tr><tr><td> Yarı kuru western blot SD electrotransfer sistemi </td><td> Bio-RAD </td><td> 170-3940 </td></tr><tr><td> At turp peroksidaz ile konjuge anti-tavşan IgG </td><td> GenScript </td><td> A00098 </td></tr><tr><td> Anti-GST tavşan poliklonal antikor </td><td> GenScript </td><td> A00097 </td></tr><tr><td> Anti-strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> Biotrace, NT nitroselüloz transferi membran </td><td> Pall Bilimsel </td><td> 27377-000 </td></tr><tr><td> Immobilon batı kemilüminesan HRP substrat </td><td> Millipore </td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

Validating farklı proteinler arasındaki etkileşimleri, moleküler düzeyde kendi biyolojik fonksiyonları incelenmesi için hayati önem taşımaktadır. Proteine ​​bağlanma in vitro ve in vivo olarak değerlendirmek için, çeşitli yöntemler vardır ve birbirlerinin eksikliklerini tamamlayacak en az iki yöntem güvenilir bilgiler elde etmek için yapılmalıdır. In vivo tayininde, iki moleküllü floresan tamamlama (BiFC) tayini, canlı hücre içerisinde protein-protein etkileşimleri algılamasını sağlar en popüler ve en az invaziv bir yaklaşımı temsil ediyor, hem de etkileşen proteinler 1,2, hücre içi lokalizasyonu belirlemek için. Bu testte, GFP veya türevleri olmayan floresan K ve C-terminal yarıya iki füzyon proteinleri test proteinler &#39;etkileşimleri nedeniyle bir araya getiren ne zaman test proteinler erimiş, ve floresan sinyal 3-6 sulandırılmış. . Çünkü sinyali Epifloresans veya konfokal mikroskobu ile kolayca tespit edilebilir, BiFC hücre biyologlar arasında seçim canlı hücreler 3 protein-protein etkileşimleri hakkında eğitim için güçlü bir araç olarak ortaya çıkmıştır. Bu testte, ancak, bazen yalancı pozitif sonuç üretebilir. Örneğin, floresan sinyal daha ziyade bu spesifik etkileşimler nedeniyle 7, küçük bir hücre içi kompartmanda ambalaj yakın nedeniyle her bildiğim kadarıyla 7 nm düzenlenmiş iki GFP parçaları ile sulandırılmış olabilir. Bu sınırlamalar nedeniyle, canlı hücre görüntüleme teknolojilerinden elde edilen sonuçlar, protein etkileşimleri tespit etmek için farklı bir prensip dayalı bağımsız bir yaklaşım tarafından teyit edilmelidir. Co-immunoprecipitation (Co-IP) ya da glutatyon transferaz (GST) açılan deneyleri, in vitro protein-protein etkileşimleri analiz etmek için yaygın olarak kullanılır gibi alternatif yöntemler temsil eder. Ancak, bu testlerin iin, ancak, test edilen proteinleri bağlama reaksiyon supportsused tampon kolayca çözünür olmalıdır. Bu nedenle, spesifik etkileşimler çözünmeyen bir protein içeren bu tekniklerin tarafından değerlendirilmelidir. Burada, biz bu zorluğu circumvents protein membran kaplama bağlayıcı testi için protokol göstermektedir. Bu teknikte, çözünür ve çözünmez proteinler arasındaki etkileşim, bir zar matriks proteinleri biri üzerinde hareketsiz çünkü güvenilir bir şekilde test edilebilir. Bu yöntem, in vivo deneyler, bu tür BiFC ile birlikte, sadakatle çözünür ve çözünmez proteinler arasındaki etkileşimleri incelemek ve karakterize etmek için güvenilir bir yaklaşım sağlar. Bu makalede, Tütün mozaik virüsü (TMV) arasında viral hücre-hücre 8-14 taşıma sırasında birden çok işlevi uygular hareket protein (MP), ve kısa bir süre önce tanımlanmış bitki hücresel interactor, tütün ankyrin tekrar içeren protein bağlama (ANK 15), bu tekniği kullanarak gösterilmiştir.

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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Test protein-protein etkileşim protein işlevsellik diseksiyon için vazgeçilmezdir. Burada, bir tanıtmak<em&gt; In vitro</emÇözünen bir protein ile bir zar hareketsiz protein prob&gt; protein-protein bağlayıcı tahlil. Bu testte, çözünmeyen bir protein ve çözüm bir protein arasındaki ilişkiyi test etmek için güvenilir bir yöntem sağlar.

Protein Membran Kaplama Testi: Çözünür ve çözünmez Proteinler Arasında Etkileşim Test Protokolü In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

22.0K Görüntüleme.  State University of New York. Test protein-protein etkileşim protein işlevsellik diseksiyon için vazgeçilmezdir. Burada, bir tanıtmak In vitro protein-protein bağlayıcı tahlil. Bu testte, çözünmeyen bir protein ve çözüm bir protein arasındaki ilişkiyi test etmek için güvenilir bir yöntem sağlar.

Video: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Membran proteinlerinin Hofstede deterjan duyarlı tespiti

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Biyokimya

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membran-OMURGA: Membran Proteinleri protein-protein etkileşimleri belirlemek için bir Biyokimyasal Aracı<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Biyomühendislik

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Bir on-membran sindirim tekniği kullanılarak protein-protein etkileşimlerinin değerlendirilmesi

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol fosfat veya Phosphoinositide etkileşim proteinleri yakınlık Kromatografi Batı Blot veya kütle spektrometresi ile bağlantılı olarak tanımlanması

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip: Protein-fosfoinositid Etkileşimlerinin Etiketsiz Bir Çalışması

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown Testi, Zorlu Protein-Protein Etkileşimlerini Test Etmek İçin Zaman Verimli Bir Araç Olarak Bakteri Hücrelerinde Ko-Ekspresyon ile Birleştiğinde

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Reseptörleri ve enzimler arasındaki işlevsel etkileşimler çalışmak için Co-immunoprecipitation tahlil

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

İmmünoloji ve Enfeksiyon

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Dayanarak protein-protein etkileşimleri ve kimliği için bir protokol<sup&gt; 15</sup&gt; K Metabolik Etiketleme, immünopresipitasyon, Kantitatif Kütle Spektrometrisi ve Affinity Modülasyon

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Biyoloji

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Genetik ve biyokimyasal Yaklaşımlar<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Protein oligomerization Değerlendirilmesi: Riyanodin Reseptör Vaka Çalışması

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies

Source: Obama, T. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/59733">Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.</a>&#160;J. Vis. Exp.&#160; (2019)
This video demonstrates co-immunoprecipitated protein complexes on a PVDF membrane for protein-protein interaction analysis. The reduced proteins from complexes were treated with trypsin to cleave the individual proteins at the smaller peptides, then, the remaining peptides were extracted from the PVDF membrane. The pulled peptide derived from both proteins in the complex was then dried and resuspended in a low concentration of formic acid for further analysis.

Etkileşim çalışmaları için birlikte immünopresipite edilmiş proteinleri hazırlamak için zar üzerinde protein sindirimi

Source: Obama, T. et al., Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.&#160;J. Vis. Exp.&#160; (2019)
This ...

Protein Membran Kaplama Testi: Çözünür ve çözünmez Proteinler Arasında Etkileşim Test Protokolü In vitro

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