Name: tandem affinity purification of protein complexes from eukaryotic cells
Uploaded: 2017-01-26T14:00:00.000+00:00
Duration: 11 min 30 s
Description: Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1. Kaplama Hücreler <ol><li> Bir gün, transfeksiyondan önce,% 10 (h / h) cenin sığır serumu (FBS) ve% 1 (h / h), penisilin / streptomisin ile takviye edilmiş Dulbecco Modified Eagle Medium (DMEM) içinde 10 mL 3.5 x 10 6 HEK293T hücreleri plaka 100 mm tabak başına (10.000 U / ml stok). Plaka 3 - Deney / durum başına 5 100 mm yemekleri yeterli protein iyileşme sağlamak için. Not: plakaların sayısı ilgili protein kompleksi ifade seviyeleri ve çözünürlüğüne bağlı olacaktır, her bir deney / durum için tespit edilmesi gerekir. Daha büyük ölçekli saflaştırma gerektiğinde, alternatif olarak, hücre çizgileri döndürücü şişelere 2 süspansiyonlar büyümeye adapte edilebilir, ancak bu yöntem, geçici nakiller için çalışmaz. </li></ol> 2. Kararlı Hücre Hatları hücrelerin transfekte ya da indükleyici <ol><li> Ertesi gün, mikroskop altında hücrelerin kontrol edin. proce önce% 80 konfluent - Hücreler 70 olmalıEding. </li><li> 100 mm tabak başına transfeksiyon reaktifi 7 plasmid DNA ug ve 21 ul toplam karışımı hücreleri transfekte. Transfeksiyon etkinliğindeki için bir kontrol olarak, bir vektör ifade GFP (Tablo 1) ile transfekt hücre. Not: doksisiklin 14, 15 karşılık olarak iki veya daha fazla kompleks alt birimleri ekspresyonunu indükleyen bir HEK293-T-Rex veya benzer sabit bir hücre doğrusu kullanılıyorsa, indükleyici hücrelere ilave edildi ve 48 saat süre ile inkübe edilmesi gerekecektir. Benzer şekilde, indükleyici ilave edilmesi üzerine GFP ekspresyonunu indükleyen bir dengeli hücre çizgisi, aynı zamanda, doğrulama için gerekli olacaktır. 2.7 adıma geçin. </li><li> 100 mm tabak başına transfeksiyon karışımı hazırlayın. 1.5 ml mikrosantrifüj tüpü içinde, (iki veya daha fazla plazmidin tekabül eder) toplam plazmid DNA 7 ug katkısız DMEM 500 ul karıştırın. Ayrı bir mikrosantrifüj tüpü içinde, katkısız DMEM wi 500 ul karışımıtransfeksiyon reaktifi inci 21 ul. Her transfeksiyon reaksiyon için tekrarlayın. </li><li> plazmid DNA çözeltisi içine transfeksiyon reaktif çözüm pipetle (ters çözüm karışmaz). en az 4 kez pipetleme karıştırın. </li><li> 10, oda sıcaklığında, bu karışım inkübe - 15 dakika, ancak artık, 15 dakika daha. </li><li> Bir medya rezervuar oluşturmak ve dikkatle hücreleri bozmadan plaka iç yüzüne damla damla transfeksiyon karışımı pipetle için plaka yatırın. orijinal düz konumuna çanak dönün ve karışım dağıtmak için hafifçe döndürün. </li><li> (Bu durum, bundan sonra &quot;doku kültür inkübatörü&quot; olarak da adlandırılır),% 5 CO2 ile nemlendirilmiş bir hücre kültürü inkübatöründe 37 ° C&#39;de inkübe hücreleri. Medya 5 saat sonrası transfeksiyon (opsiyonel) değiştirin. </li></ol> 3. kontrolü Transfeksiyon veya İndüksiyon Verimlilik <ol><li> 24 saat sonrası transfeksiyon at, bir grip altında hücrelerin görselleştirmekFlüoresan mikroskop transfeksiyon verimliliği (ya da GFP ifade kontrol HEK293T-rex istikrarlı bir hücre hattı indüksiyonu) kontrol etmek için. bir 24 saat daha inkübe edilir. </li></ol> 4. STREP Affinity saflaştırma (STREP AP) <ol><li> hücrelerin toplanması <ol><li> 48 saat sonra transfeksiyon da ortamını çıkarın ve soğuk 1 x fosfat tamponlu tuzlu su (PBS) içinde 8 ml ekleyin. plaka hücreleri ayırmak için aşağı yukarı pipet ve. </li><li> Toplayın ve 15 ml tüp içine hücre süspansiyonu aktarmak ve 4 ° C&#39;de 4 dakika boyunca 800 xg&#39;de hücreleri aşağı doğru döndürün. </li><li> PBS süpernatantı. herhangi bir artık PBS ortadan kaldırmak için (4 ° C &#39;de 1 dakika boyunca 800 x g) döndürme aşağı tekrarlayın. ileride kullanmak üzere -80 ° C&#39;de hücre pelletini Mağaza veya protein saflaştırma devam etmek için aşağıdaki prosedürü uygulayın. </li></ol></li><li> hücrelerin çözülmesi <ol><li> Pasif Lizis Tamponu hücre peleti hacmi x 5 kullanılarak hücre peletleri (~ plaka başına 250 ul) yeniden süspanse edin(PLB: 20 mM Tris-HCI pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, proteaz önleyici kokteyl tablet,% 5 h / h gliserol, ve% 1.0 NP-40) ile bir hücre süspansiyonu aktarmak 1.5 ml mikrosantrifüj tüp. </li><li> Kısaca, 4 ° C&#39;de 30 dakika süre ile süspansiyon ve nutate girdap. </li><li> 4 ° C&#39;de 10 dakika boyunca 10,000 x g&#39;de hücre süspansiyonu dönerler. </li><li> Yeni 1.5 ml mikrosantrifüj tüp içine çözünür lizatları aktarın ve hücre pelletleri atın. gibi çözülebilir lizat son hacminin% 10 Kaydet &quot;STREP AP girişi.&quot; 4 ° C&#39;de saklayın. </li></ol></li><li> STREP boncuk dengeleme NOT: İlk AP adım için kullanın STREP boncuk! STREP boncuk BAYRAK boncuklar ile aşağı çekti göre anlamlı olarak daha fazla protein kurtarmak ile kompleksler aşağı çekti. <ol><li> çıkartın (n 40 ul ×) + n% 50 STREP boncuk bulamacı ul (n = örnek sayısı) ve 4 ° C&#39;de 2 dakika süreyle 1500 xg&#39;de aşağı doğru döndürün. NOT: Geniş m kullanınpipet tanelerine outhed ipuçları. </li><li> Süpernatantı ve boncuk PLB 500 ul ekleyin. 4 ° C&#39;de 5 dakika boyunca boncuk karışımı Nutate. 4 ° C&#39;de 2 dakika boyunca 1500 x g&#39;de Spin. <ol><li> 3 yıkama toplam tekrarlayın. 40 ul x n nihai bir hacme kadar PLB boncuk tekrar. </li></ol></li><li> 4 ° C&#39;de 2 saat boyunca her bir hücre lizatı numunesi ve nutate% 50 STREP boncuk çamuru 40 ul ekle. </li></ol></li><li> STREP AP <ol><li> 4 ° C&#39;de 2 dakika süreyle 1500 xg&#39;de çözüm aşağı doğru döndürün. </li><li> 4 ° C&#39;de &quot;STREP AP Akış-through (FT)&quot; olarak supernatant ve mağaza çıkarın. </li><li> I (20 mM Tris-HCI pH 7.5, 250 mM NaCI, 1.5 mM MgCl2, 1 mM DTT,% 5 gliserol ve% 0.2 NP-40) STREP AP Yıkama Tamponu tanelere 500 ul ekle. 4 ° C&#39;de 2 dakika boyunca 1500 x g&#39;de 5, 4 ° C&#39;de dakikada santrifüj için boncuk karışımı Nutate. süpernatant atın. <ol><li> 4 yıkar toplam için tekrarlayın. <br/&gt; NOT: Dördüncü yıkamadan önce, protein arka plan azaltmak için yeni bir 1.5 ml mikrosantrifüj tüp boncuk çözüm aktarın. Bu adım gereklidir. </li></ol></li><li> 500 ul yıkama tamponu II (100 mM Tris-HCI, pH 8.0, 150 mM NaCl ve 1 mM EDTA) ilave edilir ve tüpler ters çevrilerek boncuk dengeye. 4 ° C&#39;de 2 dakika süreyle 1500 xg&#39;de Spin ve süpernatant atın. </li><li> STREP elüsyon tamponu (100 mM Tris-HCI, pH 8.0, 150 mM NaCI, 1 mM EDTA ve 2.5 mM destiobiyotin) 50 ul ilgili proteini Zehir. 4 ° C&#39;de 15 dakika boyunca 800 rpm&#39;de vorteksleyin. </li><li> 4 ° C&#39;de 1 dakika için 1500 x g&#39;de boncuk Spin. süpernatant toplayın. Bu fraksiyon &quot;STREP AP elüsyon&quot; örnek karşılık gelir. </li><li> İlk elüsyon ile elde edilen protein miktarının yarısı kadar verim olabilir ikinci elüsyon, 4 ° C &#39;de boncuk fraksiyonu kaydedin. gümüş boyama ve / veya batı blot STREP AP Tasfiye Kısım 10%16. </li></ol></li><li> BAYRAK boncuk dengeleme <ol><li> (Nx 16 ul) + n BAYRAK boncuk çözümünün ul (numune n = sayı) çıkarın ve 4 ° C&#39;de 2 dakika süreyle 1500 xg&#39;de aşağı doğru döndürün. NOT: pipet boncuk geniş ağızlı ipuçlarını kullanın. </li><li> Süpernatantı ve FLAG yıkama tamponu 500 ul ekle (100 mM Tris-HCI, pH 8.0, 150 mM NaCI, 1 mM EDTA ve% 0.1 NP-40) ile boncuklara. 4 ° C&#39;de 2 dakika boyunca 1500 x g&#39;de Spin. 3 yıkama toplam tekrarlayın. </li><li> nx 16 ul bir son hacme FLAG yıkama tamponu boncuk tekrar. </li><li> 4 ° C &#39;de geri kalan STREP AP elüsyon ve nutate gece boyunca FLAG boncuk çamuru 16 ul ekle. </li></ol></li></ol> 5. BAYRAK IP <ol><li> 4 ° C&#39;de 2 dakika boyunca 1500 x g&#39;de FLAG boncuk süspansiyonu dönerler. 4 ° C&#39;de &quot;BAYRAK IP Akış-through&quot; (FT) olarak süpernatant ve mağaza kaydedin. </li><li> 500 ekle81; çözeltiye FLAG yıkama tamponu. 4 ° C&#39;de 2 dakika boyunca 1500 x g&#39;de 5, 4 ° C&#39;de dakikada santrifüj için Nutate. 4 yıkar toplam tekrarlayın ve her yıkamadan sonra süpernatant kaldırmak. NOT: Dördüncü yıkamadan önce, arka plan azaltmak için yeni bir 1.5 ml mikrosantrifüj tüpüne protein boncuk çözüm aktarın. Bu adım gereklidir. </li><li> Final spin süpernatantı ve boncuk haline FLAG peptit 200 ng / ul içeren BAYRAK yıkama tamponu 30 uL ekleyin. 4 ° C&#39;de 2 saat boyunca 800 rpm&#39;de vorteksleyin. </li><li> 4 ° C&#39;de 2 dakika boyunca 1500 x g&#39;de süspansiyon Spin. olarak 4 ° C&#39;de supernatant ve mağaza Hasat &quot;FLAG IP Elüsyon.&quot; NOT: elüsyon numuneleri standart protokoller 16 kullanılarak gümüş leke ve / veya western blot ile teyit ve daha fazla protein deneyleri için hazır olabilir. (1) STREP AP Giriş, (2) STREP AP FT, (3) STREP AP Eluti: Batı leke kurarken, aşağıdaki örneklerin hepsi koşmak(4) BAYRAK IP FT, ve (5) BAYRAK IP Tasfiye üzerinde. Yüklenen Birimleri her bir deney için belirlenecek gerekecektir. Toplanan tüm örnekler ve boncuk kısa süreli depolama ve uzun süreli depolama için -80 ° C &#39;de 4 ° C&#39; de muhafaza edilebilir. TAP örnekleri, bileşenlerinin kimliğini doğrulamak için yeni bir post-translasyonel modifikasyonlar (PTMS) tespit etmek, ve / veya saflaştırılmış kompleksi 2, 17 ile herhangi bir yeni protein etkileşimi keşfetmek için kütle spektrometresi ile analiz edilmiştir. Bu amaçla, Coomassie jel veya gümüş leke çalıştırdıktan sonra, bantlar, temiz bir neşter kullanılarak jelden edilebilir. Kütle spektrometresi yöntemlerini tartışmak birkaç mükemmel değerlendirmeleri daha önce, 19 18 yayınlandı. </li></ol>

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The authors have nothing to disclose.

İfade ve ökaryotik hücrelerde protein komplekslerinin izolasyonu için açıklanan protokol, moleküler düzeneklerinin biyokimyasal karakterizasyonu ile sınırlı değildir, aynı zamanda işlevini düzenleyen olabilir yeni interactors ve post-translasyonel modifikasyonlar tanımlanması için de kullanılabilir. afinite etiketleri kullanımı Bu protokolde belirtilen ne ile sınırlı değildir; ancak deneyimi ilk AP adım olarak Strep kullanımı ile önemli ölçüde nihai protein-protein kompleksinin verimi art...

<html> Protein-protein etkileşimleri (PPİ) işlevleri yaklaşık 2 bilgilendirebilir biyolojik süreçler 1, bu PPI üzerinde ileri çalışmalar hassas düzenlenmesi için kritik öneme sahiptir. Çeşitli yaklaşımlar PPI çalışma ve karakterizasyonu için, ayrıca bu yeni düzenleyici protein bileşenlerinin de novo tanımlanması için geliştirilmiştir. 1989 yılında, Stanley Alanlar ve arkadaşları maya iki hibrit (Y2H) tahlil 3 bildirdi. Bu yaklaşım, Saccharomyces cerevisiae &#39;interactors oranı (yem) tanımlanmış bir protein için (avlayan) tarafsız ve kapsamlı bir tanımlanmasına izin verir. PPIs keşfetmek için dikkate değer yardımcı ek olarak, Y2H deneyi, maya hücrelerinde protein çiftleri karakterize az etkileşim alanları tanımlamak ve bu etkileşimi ortadan kaldırmak başkalaşımların teşhis edilmesi için kullanılabilir. Y2H deneyi değiştirerek, PPIs, aynı zamanda, memeli hücrelerinde incelenebilirclass = &quot;xref&quot;&gt; 4. Y2H tahlilinde (örneğin, maya, üç-hibrid sistemi) varyasyonları da protein-RNA ve hücrelerde protein küçük organik ligand etkileşimlerini incelemek için uygulanabilir. Homolog bir sistemde PPIs incelemek için bir başka genel olarak kullanılan bir araç ko-immünopresipitasyon (ko-IP) deney 5&#39;tir. ilgili bir proteini immünopresipitasyon bir antikor kullanılarak, CO-IP deney araştırmacılar, çeşitli çevre koşulları ve deneysel durumlarda hücrelere PPI izlemesine izin verir. Epitop etiketli proteinler (örneğin, FLAG, myc, Strep ve HA, diğerleri arasında) afinite temizliği (AP) yöntemleri, batı lekesi de dahil olmak üzere çok sayıda alt-tahliller için kompleks protein karışımları proteinlerin izole edilmesini kolaylaştırmıştır, gümüş leke kullanımı ve enzimatik analizi. Bununla birlikte, bu daha önceki yaklaşımların hiçbiri, in vitro bir de dahil olmak üzere daha ayrıntılı karakterizasyon için protein kompleksleri büyük miktarlarda izole edilmesini sağlamakssays, kütle spektrometresi, ve post-translasyonel modifikasyonlar tanımlanması ile düzenleyici alt birimlerin bulunması. AP yöntemin geliştirilmiş bir versiyonu, iki takip eden AP 6, 7 aracılığıyla saflaştırılır iki epitop bir füzyon proteini oluşturarak PPIs çalışmak için bir saflaştırma tekniğidir Tandem AP (TAP) olarak adlandırılır. Bu makalede, iki alt birimi farklı epitoplar ile etiketlenir ve daha sonra iki ardışık AP (STREP AP FLAG IP elde edilmiş) ile saflaştırılmış edildiği arıtma protein kompleksleri TAP yöntemin bir varyasyonu sunulmuştur. Araştırmacılar kendi ilgi protein kompleksine bunları uygulamak, böylece Biz ilk, TAP minimalist bakış (Şekil 1) ve sonra tüm deneysel adımlar ayrıntılı bir açıklama (Şekil 2) sağlarlar. TAP yöntemin uygulanabilirliğini göstermek için, (karmaşık, iyi karakterize edilmiş siklin cdk PT şu şekilde de ifade seçtikDüzenleyici alt birim, siklin T1 (CycT1) ve bir kinaz (CDK9) oluşmaktadır, ve RNA polimeraz II (pol II), 8, 9, 10 ile transkripsiyonun düzenlenmesinde görevli EFP kinaz). P-TEFb Pol II ve promoter de transkripsiyon duraklatma hafifletir ilişkili olumsuz uzama faktörleri, C-terminal sahasını fosforilleyen ve böylece transkripsiyon uzama 11, 12, 13 kolaylaştırır. Bu akılda bilinen etkileşim sayesinde, CycT1 STREP-etiketli ve FLAG-etiketli CDK9 HEK293T hücrelerinde ifade üzerindeydi. Bir karşılıklı TAP deney daha protein etkileşim kullanılan epitopların bağımsız olduğunu doğrulamak için STREP etiketli CDK9 ve FLAG-etiketli CycT1 ile gerçekleştirildi. Hücreler toplandı ve 48 saat sonra transfeksiyon parçalanmıştır. Çözünür lizat TAP ile saflaştırılmıştır (STREP AP FLAG ardındanIP). Giriş ve saflaştırılmış proteinler Western blot ve gümüş leke (Şekil 3) ile analiz edilmiştir.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH3002.2FS</td><td></td></tr><tr><td>Fetal bovine serum</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH30071</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>MP091670049</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen Laboratories</td><td>SL100688</td><td></td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11836153001</td><td></td></tr><tr><td>STREP-Tactin Superflow</td><td>IBA Lifesciences</td><td>2-1208-010</td><td></td></tr><tr><td>STREP-tag elution buffer</td><td>IBA Lifesciences</td><td>2-1000-025</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>F2426</td><td></td></tr><tr><td>Corning 100 mm &amp;times; 20 mm style dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td><td>Corning&amp;nbsp;</td><td>430167</td><td></td></tr><tr><td>Protein Lo-Bind Eppendorf</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-370</td><td></td></tr><tr><td>48 hole micro tube foam rack</td><td>Fisher Scientific</td><td>02-215-386</td><td></td></tr><tr><td>Labquake shaker rotisserie&amp;nbsp;</td><td>Thermo&amp;nbsp;</td><td>415110</td><td></td></tr></tbody></table>

tandem affinity purification of protein complexes from eukaryotic cells

Aktif protein-protein, protein-nükleik asit komplekslerinin saflaştırılması enzimsel faaliyetlerin ve yeni alt birimleri ve post-translasyonel modifikasyonlar de novo tanımlanması karakterizasyonu için çok önemlidir. Bakteriyel sistemlerde bir polipeptidler ve protein kompleksleri çok çeşitli ekspresyon ve saflaştırma için izin verir. Bununla birlikte, bu sistem, post-translasyonel modifikasyonları (örneğin, fosforilasyon ve asetilasyon) içeren protein alt birimlerinin temizleme, ve ökaryotik sisteminde ifade, sadece mevcut olan yeni düzenleyici alt birimlerin / belirlenmesini sağlamaz. Burada, ökaryotik hücrelerden geçici veya stabil ifade epitop etiketli proteinler ile protein komplekslerinin saflaştırılması kolaylaştıran bir roman, sağlam ve streptokok kullanarak verimli tandem yakınlık arıtma (TAP) yöntemiyle ve FLAG-etiketli proteinlerin ayrıntılı bir açıklama sağlar. Bu protokol, Prot karakterize uygulanabilirein karmaşık işlevsellik, kompleks alt birimlerin üzerine post-translasyonel modifikasyonlar keşfetmek ve kütle spektrometresi ile yeni düzenleyici kompleks bileşenleri tanımlamak için. Özellikle, bu TAP yöntemi ve böylece alt deney imkanları geniş bir dizi elde ökaryotik veya patojenik (viral ve bakteriyel) bileşenlerinin oluşturduğu protein kompleksleri çalışma uygulanabilir. Biz protein kompleksleri ile çalışan araştırmacılar, çok farklı şekillerde bu yaklaşımı kullanmak olabilir öneriyoruz.

Bu yazıda, (aynı zamanda P-TEFb kinaz olarak da bilinir), karmaşık iyi karakterize CycT1-CDK9 TAP yönteminin uygulanabilirliğini göstermek. Ve CDK9,-FLAG (CDK9: F) ya da CDK9-Strep (CDK9: G) ve siklin T1 bayrağı (CycT1: F): Sikline T1 Strep (s CycT1) kodlayan plazmidler (Tablo 1), HEK293T hücrelerine transfekte edildi . F ya da CycT1: (sırasıyla Şekil 3A ve B,) F ...

Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Ökaryotik Hücrelerde Protein Kompleksleri Tandem Affinity Arıtma

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

tandem-affinity-purification-protein-complexes-from-eukaryotic

Research

JoVE Journal

Biochemistry

14.9K Görüntüleme.  The University of Texas Southwestern Medical Center. We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Video: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled memeli hücrelerinden protein kompleksi olan ele geçirme kapasitesini

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Biyokimya

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Mavi Yerli PAGE ve Protein İlişkisi Profil Yöntemi ile Affinity Saf protein Kompleksler çözümleniyor

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 in 1: tek adımlı benzeşme arıtma paralel analizi için Protein-Protein ve Protein-metaboliti kompleksleri

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Kloroplast Translocon Protein kompleksleri dokunun etiketini kullanarak benzeşme arıtma

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Tandem Affinity Arıtma kullanarak Protein etkileşim Ortaklar belirlenmesi

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Biyoloji

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Protein Kompleksi belirlenmesi<em&gt; Escherichia coli</em&gt; Tandem Kütle Spektrometresi ile Kombine Sıralı Peptid Affinity Arıtma kullanarak

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL de MultiBac Protein Complex Üretim Platformu

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Protein-protein etkileşimi belirlenmesi<em&gt; Drosophila</emTandem Affinity Arıtma tarafından&gt; Adult Başlar (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Bir Tandem Affinity Etiket Yöntemiyle Yapısal Çalışmaları Yerli Komplekslerinin saflaştırılması

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Protein-Protein Etkileşimlerini İncelemek için Tandem Afinite Saflaştırma Testi

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

Ökaryotik Hücrelerde Protein Kompleksleri Tandem Affinity Arıtma

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