Biz eleştirel deneysel değerlendirme ve tartışma için UIC de O&#39;Bryan laboratuvar teşekkür ederim. Klinik ve Translasyonel Bilimler Hibe UL1TR000050 için UIC Merkezi - Bu çalışma Translational Bilim ilerlemek için GKC ve Ulusal Merkezi Amerikan Kalp Derneği Hibe 11SDG5230003 tarafından desteklenmiştir.

1. Hücre transfeksiyonu <ol><li> Transfeksiyon önce plaka hücreleri gün, immünofloresan için daha az hücre kaplama. <ol><li> Immunofloresan için: 6-plaka, kat cam kapak fişleri içinde (1 de başına) en az 4 saat süreyle 37 ° C de% 0.02 jelatin ile, orta ile bir kez yıkayın ve her bir kuyu için, plaka 1 x 10 5 HEK 293T 2 ml tam Hücreler büyüme ortamı içinde [Dulbecco&#39;nun modifiye edilmiş Eagle ortamı (DMEM) artı% 10 FBS]. </li><li> Akış sitometrik ve Western blot analizi için: levha 1,2 5 HEK 293T hücre / oyuk 6-plaka (24-çukurlu plaka başına 0,5 ml tam büyüme ortamı içinde 0.3 x 10 5 HEK 293T hücreleri içinde 2 ml tam büyüme ortamı x 10 .) </li></ol></li><li> Imalatçının protokolüne göre transfeksiyon için DNA hazırlayın: Effectene (Qiagen) immünofloresan için kullanılır. Transit-LT1 (Mirus) akım sitometrik ve Western Blot analizleri için kullanılır. Kullanılan DNA miktarı aşağıda listelenmiştir. <table border="1"><tbody><tr><td&gt; </td><td> 24 oyuklu plaka (ng) </td><td> 6-plaka (ng) </td></tr><tr><td> CFP-vektör </td><td> 10 </td><td> 50 </td></tr><tr><td> VN N-AKAP-Lbc </td><td> 200 </td><td> 1.000 </td></tr><tr><td> VC N-PDE4D3-FL (tam boy PDE4D3) </td><td> 2.5, 5, 10, 20, 40, 80 </td><td> 100 </td></tr><tr><td> VC N-PDE4D3-UCR1 (UCR1 etki alanı içeren bir PDE4D3 N-terminal parçası) </td><td></td><td> 100 </td></tr><tr><td> VC N-PDE4D3-UCR2 + CAT (UCR2 ve katalitik etki içeren bir PDE4D3 C-terminal parçası) </td><td></td><td> 100 </td></tr></tbody></table> Transfeksiyon Effectene (6-de) kullanılarak: <ol><li> Oda sıcaklığında 5 dakika boyunca belirtilen plazmid DNA + miktarı 4 ul artırıcı 100 ul tampon EM için, karışım, ve inkübe ekleyin. </li><li> 10 dakika boyunca 5 ul Effectene, karışım, ve inkübe eklemeoda sıcaklığı. Ekle hücrelere bırak-bilge. </li></ol> : Transit-LT1 (6-well/24-well) kullanarak transfeksiyon <ol><li> Bir steril tüp içine Opti-MEM I serumsuz ortamda 250 μl/50 ul plasmid DNA Belirtilen miktarlarda ekleyin. </li><li> Seyreltilmiş DNA karışıma Transit-LT1 reaktif ekleyin ve karıştırmak için hafifçe pipetle. (Transit-LT1 Reaktif: DNA oranı DNA 1 mikrogram başına Transit-LT1 reaktif 3 ul). </li><li> Oda sıcaklığında 30 dakika süreyle inkübe edilir ve hücreleri damla damla ilave edin. </li></ol></li></ol> 2. Sitometrisi, Western Blot, ve İmmünofloresan için Hücre Hazırlık <ol><li> 24 saat sonrası transfeksiyon, hücreler hasat öncesi Epifloresans mikroskop altında floresan kontrol edin. </li><li> Akım sitometri analizi için hücre hazırlama: <ol><li> 24-well/6-well plakaları ile, 0.5 ml / 2 ml buz gibi soğuk PBS ile bir hücreleri yıkayın. </li><li> 50 μl/250 ul% 0.05 tripsin kullanarak hücreleri ayırmak. </li><li> 200 ul / 1 ml DMEM ortamı ile nötralize tripsin. </li><li> Co5 dakika boyunca 500 x g&#39;de santrifüj ile llect 250 ul hücreleri (her ikisi de 24 oyuklu ve 6-delikli plaka için), Western blot analizi için geriye kalan hücreler (1 mi) kullanın. </li><li> 250 ul FACS buffer içinde yeniden süspanse hücreleri [PBS +% 0.5 (w / v) BSA, 2 mM EDTA +] akış sitometri analizi için buz üzerinde depolar. Akım sitometri analizi hemen olmayacak eğer hücreler (PBS içinde)% 1 paraformaldehid sabitlenebilir. </li></ol></li><li> Western Blot analizi için hücre hazırlama: akım sitometri analizi için hücre hazırlama paralel olarak gerçekleştirdi. <ol><li> 2 ml buz gibi soğuk PBS ile yıkayın 1x hücreleri, hücre parçalama tamponu 100 ul [10 mM sodyum fosfat tamponu, pH 6.95, 150 mM NaCI, 5 mM EDTA, 5 mM EGTA,% 1 Triton X-100] eklenerek hücre parçalandığı . </li><li> 4 de 21.000 xg&#39;de 10 dakika boyunca santrifüj ile hücre lizatı toplamak ° C </li><li> , Bradford protein testi ile protein konsantrasyonu ölçmek SDS-PAGE için şerit başına protein eşit miktarda yük ve immün için nitroselüloz transfer. </li></ol&gt; </li><li> Immünofloresan için hücre hazırlama. <ol><li> 2 ml PBS ile durulayın 3x hücreler, oda sıcaklığında 10 dakika boyunca% 3.7 paraformaldehid (PBS) 1 ml hücre düzeltmek için, daha sonra 5 dakika boyunca 2 ml PBS ile 3 kez yıka. </li><li> Montaj orta kullanarak, bir cam slayt üzerinde kayma kapağı monte edin. Gerekirse oje kullanarak lamel kenarları mühür. </li><li> Muayene ° C önce 4 de slaytlar saklayın. </li></ol></li></ol> 3. Akım Sitometri ve İmmünofloresan Mikroskopi <ol><li> Flow sitometri bir Beckman Coulter Mavi 488 nm ile donatılmış ADP, 405 nm ve 642 nm katı hal lazerleri kullanılarak yapılır. Zirve yazılım satın alma ve analiz için kullanılır. <ol><li> Standart boncuklar kullanarak akış sitometresinin kalibre. </li><li> Yolluk canlı hücre popülasyonu için doğrusal bir ölçekte Arsa İleri dağılım (FSC) / yana dağılım (SSC). </li><li> Yolluk olmayan toplu canlı hücre popülasyonu için arsa gerilim / FSC darbe genişliği. </li><li> Arsa count/FL6 (OBP fluorescencyolluk olmayan toplu canlı CFP-pozitif hücreler için bir günlük ölçekte e, 405 nm). </li><li> BiFC yoğunluğu için bir günlük ölçekte Arsa count/FL1 (YFP floresan, 488 nm). </li><li> FSC, SSC, FL1 ve FL6 fotoğraf çoğaltıcı tüp (PMT) Her bir sinyal için eşik ayarlamak için voltaj ayarlamak, YFP-CFP-çift negatif örnekleri çalıştırın. </li><li> Run (hem YFP + ve CFP +) için örnekler gelecek off-line tazminat pozitif ve tek </li><li> Analiz için en azından 10,000 kapılı olayları (olmayan toplanan canlı CFP + hücreleri) elde edin. </li></ol></li><li> . İmmünofloresan mikroskobu bir Zeiss LSM 510 konfokal mikroskop Not kullanılarak yapılır: tüm ayarlar arasında tutarlı (örneğin, zum, iğne deliği, dedektör kazanç, amplifikatör ofset, çerçeve boyutu, tarama hızı, tarama ortalama ve lazer güç olarak) tutmak önemlidir tüm verilerin uygun bir karşılaştırma için örnekler. </li></ol> 4. Veri Analizi (Summit Yazılımı kullanarak Yapılan) <ol><li> Acquisit için olana benzer bir analiz protokolü ayarlamauygun yolluk ile iyon: Canlı hücreler için FSC / SSC nokta arsa Gate 1. Olmayan toplu canlı hücreler için Gate 1 FSC / darbe genişliği Kapısı 2. Olmayan toplu OBP + canlı hücreler için Gate 3 sayısı / CFP Gate 3. </li><li> YFP ve YFP + ve OBP + tek pozitif hücreleri kullanarak OBP sinyal telafi. </li><li> CFP / YFP pozitif hücreler için yeniden belirlemek kesme hatları. </li><li> İhracat YFP CFP floresan yoğunluğu (MFI) + veri komplo için Excel&#39;e hücreler anlamına gelir. </li><li> AKAP-Lbc, PDE4D3 ifade seviyelerine YFP MFI Normale ve Western blot tarafından belirlenen, α-tubulin yükleme kontrolü. </li></ol>

<ol>
	<li>Collura, V., Boissy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+protein-protein+complexes+to+interactomics.">From protein-protein complexes to interactomics.</a> Subcell Biochem. 43, 135-183 (2007).</li><li>Stumpf, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).</li><li>Shekhawat, S. S., Ghosh, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-protein+systems:+beyond+binary+protein-protein+interactions.">Split-protein systems: beyond binary protein-protein interactions.</a> Curr. Opin. Chem. Biol. 15, 789-797 (2011).</li><li>Dwane, S., Kiely, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+used+to+study+how+protein+complexes+are+assembled+in+signaling+cascades.">Tools used to study how protein complexes are assembled in signaling cascades.</a> Bioeng. Bugs. 2, 247-259 (2011).</li><li>Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+measuring+protein+interactions+in+vivo+and+in+vitro.">New methodologies for measuring protein interactions in vivo and in vitro.</a> Curr. Opin. Struct. Biol. 15, 4-14 (2005).</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., Suarez, C. D., Hu, C. D. <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+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nat. Protoc. 3, 1693-1702 (2008).</li><li>Wong, K. A., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation.">Bimolecular fluorescence complementation.</a> J. Vis. Exp. (50), e2643 (2011).</li><li>Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+general+principles+and+applications+to+selected+studies+in+tumor+biology.">Flow cytometry: general principles and applications to selected studies in tumor biology.</a> Int. Adv. Surg. Oncol. 2, 125-153 (1979).</li><li>Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+GLUT4+translocation+to+the+plasma+membrane+by+flow+cytometry.">Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry.</a> J. Vis. Exp. (45), e2429 (2010).</li><li>Smith, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+confocal+microscopy.">Basic confocal microscopy.</a> Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).</li><li>Westwick, J. K., Lamerdin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+discovery+with+contextual+assays+and+cellular+systems+analysis.">Improving drug discovery with contextual assays and cellular systems analysis.</a> Methods Mol. Biol. 756, 61-73 (2011).</li><li>Kilpatrick, L. E., Holliday, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+pharmacology+of+G+protein-coupled+receptor+signaling+complexes+using+bimolecular+fluorescence+complementation.">Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.</a> Methods Mol. Biol. 897, 109-138 (2012).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

BiFC protein-protein etkileşimleri incelemek için basit ve hassas bir yöntemdir. Bu yöntem, yeni bir protein-protein etkileşimleri tanımlamak için kullanılamaz, ancak, bu hücre içinde, protein-protein etkileşimi teyit etmek için ve fonksiyonel özelliklerini incelemek için özellikle uygundur, örneğin protein-protein etkileşimi siteleri eşleme protein kompleksleri hücre altı konumlanması olarak, ve protein-protein etkileşimi modüle eden küçük moleküller / peptitlerin taranması için. VN ve VC ...

650.000 protein-protein etkileşimleri, normal hücre fonksiyonlarını 1,2 korunmasında kritik rol oynayan, insan interactome var olduğu tahmin edilmektedir. Ortak immunoprecipitation yanı sıra (eş-IP), altın standart (PCA) hücreleri 3 içinde protein-protein etkileşimleri tespit duyarlılığını artırmak için geliştirilmiştir, hücre lizat birkaç protein parçası tamamlama deneyleri protein-protein etkileşimleri incelemek için. Teknikleri Förster rezonans enerji transferi (FRET), Biyoparlaklık Rezonans Enerji Transferi (BRET) ve Bimoleküler Floresan tamamlama (BiFC) 4,5 içerir. Her potansiyel bir etkileşim protein ortağı (bu örnekte biz AKAP-Lbc ve PDE4D3 kullanın) sigortalarla, BiFC bir floresan protein iki parça (bir YFP varyant burada Venüs kullanın) kolaylaştırdı dernek dayanmaktadır. F tarafından görüntülenmiştir olabilir fonksiyonel floresan hücreleri sonuçları içinde ilgi iki protein, etkileşimiCanlı veya sabit hücreleri 6,7,8 kullanarak luorescence mikroskobu. Diğer PCA ile karşılaştırıldığında, BiFC canlı hücrelerde hücresel yerleşimi çalışma potansiyeli olan, duyarlı ve daha az teknik zordur. Bu tekniğin büyük bir dezavantajı, ancak bir kere oluştuktan sonra, floresan protein kompleksi geri alınamaz olmasıdır. Bu nedenle dinamik protein-protein etkileşimleri incelemek için iyi bir yöntem değildir. Bu yazıda, Venüs floresan yoğunlukları izleyerek AKAP-Lbc ile PDE4D3 etkileşimi siteleri eşleştirmek için BiFC kullanın. (Otomatik yüksek kapasiteli makineler kullanılarak gerçekleştirilmiştir değilse) zaman alıcı ve emek-yoğun floresan mikroskobu kullanarak geleneksel analizi ile karşılaştırıldığında, akım sitometri, kısa bir süre 9 üzerinde heterojen nüfus hücrelerin binlerce basit bir kantitatif analiz sağlar 10. Burada, akış sitometrik-BiFC analizi ve ortak IP geleneksel bir yan-yana karşılaştırma yaparak, biz göstermek bu iki methods karşılaştırılabilir veri sağlamak, ancak, akış sitometrik BiFC analizi zaman alıcı az ve ilgi hücreleri sınırlı zaman daha yararlı olabilir daha az malzeme kullanır.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Dulbecco&amp;#8217;s modified Eagle&amp;#8217;s media (DMEM)</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>Penicilin-Streptomycin (pen/strep), 100x</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>Anti-Flag antibody</td><td>Sigma</td><td>M2, F1804</td><td></td></tr><tr><td>Anti-HA antibody</td><td>Covance</td><td>16B12, MMS-101R</td><td></td></tr><tr><td>&amp;#945;-tubulin</td><td>Sigma</td><td>DM1A, T9026</td><td></td></tr><tr><td>Anti-GFP antibody</td><td>Clontech</td><td>632569</td><td></td></tr><tr><td>Cell culture plates, 6-well tissue culture treated</td><td>Thermo Fisher Scientific</td><td>130184</td><td></td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>Maxiprep plasmid purification kit, high speed</td><td>Qiagen</td><td>12663</td><td></td></tr><tr><td>Dulbecco&amp;#8217;s Phosphate-buffered saline (DPBS), sterile 1x</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Trypsin-EDTA, 0.05% (w/v)</td><td>Gibco</td><td>25300</td><td></td></tr><tr><td>Polystyrene round-bottom tubes for FACS staining</td><td>BD Biosciences</td><td>352052</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>S74337MF</td><td></td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Invitrogen</td><td>P-36931</td><td></td></tr><tr><td>Superfrost plus Microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Fisherfinest premium cover glass</td><td>Fisher Scientific</td><td>12-548-5P</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; air-jacketed Incubator</td><td>NuAIR DH autoflow</td><td></td><td></td></tr><tr><td>Confocal microscope LSM510 META</td><td>Carl Zeiss, Inc</td><td></td><td></td></tr><tr><td>Electrophoresis and transfer unit</td><td>Biorad</td><td></td><td></td></tr><tr><td>Cyan ADP Flow Cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

Hücrelerin içinde protein-protein etkileşimleri incelemek için yöntemler arasında, Bimoleküler Floresan tamamlama (BiFC) oldukça basit ve hassastır. BiFC bir floresan protein olmayan iki floresan parçalarının (venüs, bir sarı floresan protein varyantı, burada kullanılan) kullanılarak floresan üretimi esas alınmaktadır. Floresan olmayan Venüs parçaları (VN ve VC) bağlı VN-AKAP-Lbc-VC-PDE4D3 etkileşim ve fonksiyonel bir floresan protein oluşumu için floresan verecek şekilde iki etkileşen proteinleri (bu durumda, AKAP-Lbc ve PDE4D3) kaynaşmıştır hücrelerin içinde. BiFC protein komplekslerinin hücre altı konumlanması ve floresan yoğunluğuna dayalı protein etkileşimleri gücü hakkında bilgi sağlar. Bununla birlikte, protein-protein etkileşimi kuvvetini ölçmek için mikroskopi kullanılarak BiFC analizi zaman alıcı ve protein ekspresyonu ve etkileşim içinde heterojenite nedeniyle biraz görecelidir. Sitometrik akış kaplinBiFC metodolojisi ile analizi, floresan BiFC protein-protein etkileşimi sinyalini doğru olarak kısa bir süre içinde hücrelerin büyük bir miktar için ölçülebilir. Burada, AKAP-Lbc ile etkileşim için gerekli olan PDE4D3 bölgeleri eşleştirmek için bu metodolojinin bir uygulama göstermektedir. Bu yüksek kapasiteli yöntem, protein-protein etkileşimleri düzenleyen tarama faktörlere uygulanabilir.

AKAP-Lbc ve PDE4D3 yapıları (Şekil 1B) sırasıyla, VN ve VC parçaları erimiş edildi. AKAP-Lbc ve fonksiyonel YFP floresan (Şekil 1A) içinde PDE4D3 sonuç etkileşimi. Burada PDE4D3 içinde haritası AKAP-Lbc bağlayıcı sitelere BiFC yöntemini kullanın. Memba korunmuş bölge 1 (UCR1), yukarı korunmuş bölge 2 (UCR2) ve katalitik bölgesi (CAT) PDE4D3 bağlama sitesi tarama için kullanılan bu nedenle, tam boy (FL), UCR1 ve UCR2 artı CAT, PDE4 aile proteinler arasında k...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Bimoleküler Floresan tamamlama akış sitometrik analizi protein-protein etkileşimleri incelemek için bir yüksek kapasiteli nicel yöntem sağlar. Bu metodoloji, eşleme protein bağlama siteleri ve protein-protein etkileşimleri düzenleyen faktörler taranması için uygulanabilir.

Bimoleküler Floresan tamamlama akışı Sitometrik Analizi: Protein-protein etkileşim Eğitim Bir Yüksek Verimli Kantitatif Yöntemi

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

flow-cytometric-analysis-bimolecular-fluorescence-complementation

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.3K Görüntüleme.  University of Illinois at Chicago. Bimoleküler Floresan tamamlama akış sitometrik analizi protein-protein etkileşimleri incelemek için bir yüksek kapasiteli nicel yöntem sağlar. Bu metodoloji, eşleme protein bağlama siteleri ve protein-protein etkileşimleri düzenleyen faktörler taranması için uygulanabilir.

Video: Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Bir floresan dalgalanma spektroskopisi tahlil Protein-Protein etkileşimlerinin hücre-hücre kişiler

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Biyokimya

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Bimoleküler Floresan tamamlama ile fotoaktive Yerelleştirme Mikroskobu (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Biyomühendislik

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Yakınlık Ligasyon Tayini kullanılarak C. elegans Germline Ribonükleoprotein Kompleksi Montaj Yerinde Algılama

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Gelişim Biyolojisi

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Bimoleküler Floresan tamamlama

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Biyoloji

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Bimolecular tamamlayıcı benzeşme arıtma (BiCAP) tarafından parçalanmış çok protein sinyal kompleksleri

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

İmmünoloji ve Enfeksiyon

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Ağ Geçidi Uyumlu Bimoleküler Floresan tamamlama (BiFC) kullanarak Bitki Protein Etkileşimleri Tespiti

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Algılama ve ölçme protein Self-montaj In vivo tarafından akış sitometri

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria

Source: Manko, H. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61602">FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.</a> J. Vis. Exp. (2020)
The video describes the FLIM-FRET imaging technique to determine the protein-protein interaction in live bacteria expressing cytoplasmic proteins labeled with fluorescent proteins, a donor eGFP, and acceptor mCherry. The combined technique also allows the quantification of the interacting proteins.

Canlı Bakterilerde Protein-Protein Etkileşimlerinin Karakterizasyonu için FLIM-FRET Görüntüleme

Source: Manko, H. et al., FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. J. Vis. Exp. (2020)
The video describes the FLIM-FRET ...

JoVE Encyclopedia of Experiments

Biyolojik Teknikler

Biyomoleküler Etkileşim Tespit Teknikleri

Protein-protein

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

Source: Nickerson, A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53154">Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM).</a> J. Vis. Exp. (2015)
This video describes BiFC-PALM &#8213; a combination of photoactivated localization microscopy and bimolecular fluorescence complementation &#8213; to assess protein-protein interactions. BiFC involves the fusion of two fluorescent protein fragments with two interacting proteins of interest. When the two proteins interact, the fragments are brought into proximity, which allows the two parts to come together and reconstitute a functional fluorescent protein. The fluorescence of a single fluorophore is localized at a single molecule level inside cells by PALM.

Bimoleküler Floresan Tamamlama-Birleştirilmiş Fotoaktif Lokalizasyon Mikroskobu

Source: Nickerson, A. et al., Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM). J. Vis. Exp. (2015)
This ...