Name: visualization of protein-protein interaction in nuclear and cytoplasmic fractions by co-immunoprecipitation and in situ proximity ligation assay
Uploaded: 2017-01-16T15:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

This study was supported by the Swiss National Science Foundation (SNSF, 31003A_163305).

1. ko-immünopresipitasyon <ol><li> Tohum bir 6-yuvalı plaka içindeki oyuk başına 2 x 10 5 hücre HEK293 hücreleri 2 mi,% 10 fetal sığır serumu (FBS) ile Dulbecco Modifiye Kartal Ortamı (DMEM) ilave 100 U / ml penisilin-streptomisin (LTR yükseltici) durumunda . </li><li> % 5 CO2 ile 37 ° C &#39;de 48 saat boyunca hücrelerin büyütün. </li><li> soğukluğunda fosfat tamponlu tuzlu su (PBS) ile üç kez hücreleri yıkayın. 4 ° C&#39;de 5 dakika boyunca 500 x g&#39;de santrifüj ile hasat hücreleri. </li><li> ticari nükleer ve sitoplazmik çıkarma reaktifler kullanılarak nükleer ve sitoplazmik kesirler hazırlayın. bazı değişikliklerle üreticinin talimatı uygulayın. <ol><li> Bir ko-immünopresipitasyon deneyi (bir 6-yuvalı plaka 3 kuyu ile yaklaşık olarak% 90 oranında konfluent) 3 x 10 6 hücre kullanın. en yüksek hızda 15 saniye için 300 ul soğuk sitoplazmik ekstraksiyon çözeltisi (CER I) ve vorteks ekleyin. </li><li> Buz üzerinde 30 dakika süreyle inkübe edin. İnkübasyon, vorte sırasında5 s her 10 dakika için, x. </li><li> 5 saniye boyunca 16,5 ul soğuk sitoplazmik ekstraksiyon çözeltisi II (CER II), girdap ekleyin ve buz üzerinde 5 dakika boyunca inkübe edin. </li><li> 4 ° C&#39;de 5 dakika boyunca 16,000 x g&#39;de 5 saniye ve santrifüj vorteksleyin. </li><li> Yeni bir ön-soğutulmuş tüp içine süpernatant (sitoplazmik ekstresi) aktarın ve kullanılıncaya kadar buz üzerinde tutmak. </li><li> Her zaman yukarı pipetleme ve beş kat aşağı 1 ml soğuk PBS ile çözülmez topakları (içeren çekirdekleri) üç kez yıkayın. yıkamadan sonra PBS kaldırmak. </li><li> 15 saniye süre ile 150 ul soğuk çekirdek ekstraksiyon çözeltisi (EŞ), girdap ekleyin ve buz üzerinde 1 saat inkübe edilir. İnkübasyon sırasında, 15 saniye ve pipet 200 ul ucu ile 10 kez, her 10 dakika boyunca vorteks. </li><li> 4 ° C&#39;de 10 dakika boyunca 16,000 x g&#39;de 15 saniye vorteksleyin ve santrifüjlenir. </li><li> Yeni bir ön-soğutulmuş tüp içine süpernatant (nukleer extract) transfer kullanana kadar buz üzerinde tutmak. </li></ol></li><li> Her girdi olarak nükleer ve sitoplazmik özler hacminin% 10 çıkar. </li><li> bir1 ml&#39;lik bir son hacme kadar geri kalan özler, soğuk PBS dd. Buz üzerinde kullanıma kadar muhafaza edin. Not: Protein G-konjuge manyetik boncuklar kullanıldığında Ön temizleme gerekli değildir. Arka plan yüksek, ancak, 40 ul protein G-konjuge manyetik boncuk (% 50 bulamaç) ve, bir döndürücü üzerinde 30 dakika boyunca 4 ° C&#39;de bu aşama lizat seyreltilmiş 1 ml inkübe edilerek ön temizleme yapar. Manyetik raf ile boncuklar ayrı süpernatant (önceden temizlenmiş lizat). boncuk atın. </li><li> Protein G-konjuge manyetik boncuklar ile çift primer antikor için, 4 ug tavşan poliklonal anti-RBM3 antikoru veya tavşan IgG (negatif kontrol), 200 ul PBS artı Tween 20 tamponu ile Protein G-konjuge manyetik boncuk (% 50 çamurlu) ul 40 inkübe (PBST,% 0.02 Tween 20) ile, bir döndürücü üzerinde 40 dakika boyunca oda sıcaklığında (RT). </li><li> Ayrı bir manyetik raf ile boncuk ve süpernatant antikor birleştiğinde, ve supernatant atın. 200 ul PBST (% 0.02) ile bir kez boncuk yıkayın. </li><li> 1 eklemi tanelere lizatları (ya da gerektiğinde, önceden temizlenmiş lizatları halinde) ile seyreltildi ve gece boyunca 4 ° C &#39;de, bir döndürücü üzerinde inkübe edin. </li><li> Ertesi gün, bir manyetik raf boncuk ve süpernatant ayırın ve supernatant atın. 20 rpm sabit bir hızla, bir döndürücüde 4 ° C de, her bir tüp için 0.5 mi PBST (% 0.02) ile 3 x 10 dakika yıkanır. </li><li> 40 ul numune tamponu (1 x ticari numune tamponu ve 50 mM 1,4-ditiyotreitol (DTT)) eklenerek boncuk proteinleri Zehir. 10 dakika boyunca 70 ° C&#39;de ısıtın. Yeni 1.5 ml tüp aşağı Spin ve taşıyıcı sıvılar. </li><li> 10 dakika boyunca 70 ° C&#39;de ve ısı (1 x ticari numune tamponu ve 50 mM DTT nihai konsantrasyon) numune tampon maddesi girdi lizatları seyreltilir. Bir prekast% 4-12 bis-Tris jel son adımdaki yük giriş ve immunoprecipitated proteinler. Her çukurun yükleme hacmi 20 ul aşmamalıdır. 35 dakika boyunca buz üzerinde 1 x ticari çalışan tamponu içinde elektroforez gerçekleştirin. NOT: Yük nükleer ve sitoplazmik özühücrelerin aynı başlangıç ​​miktarını yansıtır 2 (v / v), 1 bir oranda s. </li><li> 4 ° C&#39;de 2 saat süre ile 30 V 1x ticari transfer tampon maddesi içinde PVDF membrana transfer proteinleri. </li><li> % 5 blok membran bir karıştırıcıda 40 dakika boyunca oda sıcaklığında PBST süt (% 0.1 Tween 20) yağsız. </li><li> gece boyunca 4 ° C&#39;de PBST (% 0.1): Primer antikor ile membran (1000 de 1 seyreltilmiş) inkübe edin. </li><li> bir çalkalayıcı üzerinde oda sıcaklığında PBST (% 0.1) ile 3 x 10 dakika yıkanır. </li><li> 1 saat boyunca oda sıcaklığında PBST (% 0.1): (5.000 iki 1&#39;de seyreltilmiş) yaban turpu peroksidaz (HRP) ile membran inkübe anti-fare ya da anti-tavşan sekonder antikor ile konjüge edilmiş. </li><li> bir çalkalayıcı üzerinde oda sıcaklığında PBST (% 0.1) ile 3 x 10 dakika yıkanır. </li><li> Cm2 zar başına 0.2 mi zenginleştirilmiş kimyasal aydınlatma (ECL) substrat karışımı kullanarak, 5 dakika boyunca oda sıcaklığında inkübe edilir. kırmızı ışık hariç itibaren bu adımdan tüm ışık kaçının. </li><li> Sıvı atın ve bir film kaseti içinde bir X-ışını filmine maruz kalmaktadır. Bir otomatik film pr kullanarak filmi geliştirmekocessing makinesi. </li></ol> 2. İmmünositokimya ve Proximity ligasyon Testi <ol><li> Tohum 0.4 ml DMEM içinde bir 8 bölmeli poli-D-lizin kaplı slayt bölme başına 1,5 x 10 4 hücre HEK293 hücreleri,% 10 FBS ve 100 U / ml Pen-Strep ile takviye edilmiştir. </li><li> % 5 CO2 ile 37 ° C &#39;de 48 saat boyunca hücrelerin büyütün. </li><li> Aspire ortamı ve oda sıcaklığında 10 dakika süreyle% 4 paraformaldehid (PFA) ile hücrelerin tespit. </li><li> Aspire PFA ve odasının başına 0.5 ml PBS ile yıkama 3 x 10 dk. </li><li> Permeabilize ve 1 saat oda sıcaklığında PBS içinde% 0.5 Triton X-100 ve% 5 normal keçi serumu (NGS) blok hücreleri. </li><li> gece boyunca 4 ° C&#39;de bir çalkalayıcı üzerinde PBS içinde% 0.1 Triton X-100 ve% 5 NGS primer antikorlar ile inkübe edin. Fare monoklonal anti-NF90 antikoru ve tavşan poliklonal anti-RBM3 antikoru 1 seyreltin: PBS içinde 100 çift boyama. Negatif kontrol için, bağımsız bir şekilde, iki birincil antikor ya da ihmal ve üçüncü kontrol antikorları sayın. NOT: Ticari engelleme ve seyreltilmesi reaktifleri kullanmayın. </li><li> bölme başına 0.5 ml PBS ile 3 x 10 dakika yıkanır. </li><li> Immünsitokimya veya yakınlık ligasyon tahlil protokolleri tabi <ol><li> İmmünositokimya <ol><li> 1 ile inkübe: 500 seyreltilmiş, yeşil fluoresan boya-bağlı anti-fare ve kırmızı fluoresan boya-bağlı anti-tavşan sekonder antikor, oda sıcaklığında 1 saat karıştırıldı. itibaren bu aşamada ışık kaçının. </li><li> 10 dakika boyunca oda sıcaklığında PBS içinde 5000: 4 &#39;, 6-diamidin-2-fenilindol (DAPI) ile karşıt çekirdekler 1 seyreltilmiştir. </li><li> bölme başına 0.5 ml PBS ile 3 x 10 dakika yıkanır. </li><li> cam slayt ve kuru gelen odaları çıkarın. slayt başına orta montaj, 250 mikronun ile monte edin. </li></ol></li><li> Yakınlık ligasyon tahlili <ol><li> yakınlık ligasyonu tahlil probları hazırlayın. Karıştır ve th lige farklı oligonükleotidler ile bağlı iki proksimite ligasyonu deney probları (anti-fare ve anti-tavşan sekonder antikor seyreltikBir 8 bölmeli slayt (cm2 başına yaklaşık 40 ul 320 ul toplam hacimde PBS içinde% 0.1 Triton X-100, 5 ile% 5 NGS): iki ligasyon çözeltisi içinde oligonükleotit), her iki 1 kaba ilaveli . 20 dakika boyunca oda sıcaklığında inkübe edin. </li><li> cam slayt odaları çıkarın ve sulandırılmış probları ekleyin. 1 saat boyunca 37 ° C&#39;de nemli bir inkübatörde inkübe edin. </li><li> ligasyon çözeltisi hazırlayın. 8 ligaz ul karıştırın 64 ul 5 x ligasyonu stok ve 248 ul H 2 O </li><li> slayt sıvıyı dokunun ve 1x Yıkama Tampon A 2 x 5 dakika yıkayın (takımla birlikte verilmektedir). </li><li> Ligasyon çözeltisi ilave edin ve 30 dakika boyunca 37 ° C&#39;de nemli bir inkübatörde inkübe. </li><li> amplifikasyon çözüm hazırlayın. Itibaren bu aşamada ışık kaçının 4 ul polimeraz, 64 ul 5x büyütme stok ve 252 ul H 2 O karıştırın. </li><li> slayt sıvıyı dokunun ve 1x Yıkama Tamponu A&#39;da 2 x 2 dk yıkama </li><li> amp eklelification çözeltisi ve 100 dakika için 37 ° C &#39;de karanlık bir nem inkübatöründe inkübe edin. </li><li> slayt sıvıyı dokunun ve 1x Yıkama Tamponu B 2 x 10 dakika yıkayın (takımla birlikte verilmektedir). 0.01x Yıkama Tamponu B&#39;de 1 dakika yıkayın </li><li> slayt kurutun ve slayt başına (DAPI ile) ticari bir montaj orta 250 ul monte edin. </li></ol></li></ol></li><li> 10X mercek lens ve 20X objektif lens kullanarak bir mikroskop altında floresan sinyalleri inceleyin. CCD kamera ile görüntüler elde. </li></ol>

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The authors have nothing to disclose.

Her iki yöntem için çeşitli faydalar yanı sıra eksiklikler vardır. nispeten yeni bir teknik olarak, yakınlık ligasyon tahlili açık bir avantajı, tek hücre düzeyinde yerine heterojen bir hücre yığın protein-protein etkileşimlerini aydınlatmak için fizibilite olup. Yüksek büyüklükte ve (konfokal mikroskop ile) çözünürlükte Görüntüler tek floresan noktalar sayarak ölçümü için olanak sağlamaktadır. Buna karşılık, Western Blot ile ko-immünopresipitasyon tekniğinin gelenek...

Nükleer faktör 90 (NF90) interlökin-2 sonrası transkripsiyonu ve miRNA biogenez 1-3 düzenlenmesi viral enfeksiyona yanıt, yönetmelik dahil olmak üzere sayısız fonksiyonları ile çok izoformu proteindir. RBM3 bir RNA bağlayıcı protein, çeviri ve miRNA biyogenezine katılan ve hipotermi ve hipoksi 4-6 dahil olmak üzere çeşitli stres tarafından uyarılan edilebilir. Son zamanlarda, bir protein kompleksi 7 NF90 ve RBM3 bulundu. NF90 ve RBM3 etkileşimi katlanmamış bir protein tepki 7 protein kinaz RNA gibi endoplazmik retikulum kinaz (PERK) aktivitesini modüle etmek için gereklidir. NF90 ve RBM3 Hem çekirdekte ama sitoplazma içine küçük bir NF90 oranı ve RBM3 mekik ağırlıklı olarak bulunduğu ve PERK aktiviteyi düzenleyen, özel fonksiyonlar için birbirlerine orada örneğin bağlama vardır. Nedenle, yeni gösterebilir, hücre içi bölmeye NF90-RBM3 etkileşimleri dağılımını görselleştirmek için önemlidirİlgili bölmeye onların çeşitli rolleri. Yıllar önce, maya iki hibrid (Y2H) iki protein 8 arasındaki etkileşimi saptamak için geliştirilmiştir. Bununla birlikte, erimiş proteinleri yapay bir yapı için, yanlış pozitif sonuçlar, bu yöntemin uygulanmasını sınırlamıştır. Uzun bir süre, ko-immünopresipitasyon özellikle endojen koşullarda 9, protein-protein etkileşimleri analiz etmek ana tekniktir. Süper hassasiyet ve kesinlik arzu edildiğinde kütle spektrometresi kullanılır ise birlikte imüno-çökeltilmiştir protein kompleksi analiz etmek için, Batı benek, en uygun bir tekniktir. Son yıllarda, proksimite ligasyonu deney yerinde 10,11 hem hücre ve dokularda protein-protein etkileşimlerini tespit etmek için yeni bir yöntem olarak geliştirilmiştir. Burada, NF9 yakalayan en popüler ko-immünopresipitasyon yöntemi ve nispeten yeni yakınlık ligasyonu tahlil yöntemi karşılaştırıldısub-hücresel fraksiyonlarında, 0 RBM3 etkileşimi. Biz de avantajları ve her iki tekniğin sınırlamaları tartışılmıştır.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Sigma</td><td>D6429</td><td>High glucose&lt;br/&gt; 4,500 mg/L</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Thermo Fisher Scientific</td><td>10270106</td><td></td></tr><tr><td>Penicillin-Streptomycin (PenStrep)</td><td>BioConcept</td><td>4-01F00-H</td><td></td></tr><tr><td>NE-PER Nuclear and Cytoplasmic Extraction Reagents</td><td>Thermo Fisher Scientific</td><td>78833</td><td></td></tr><tr><td>1,4-Dithiothreitol (DTT)</td><td>Carl Roth</td><td>6908.3</td><td></td></tr><tr><td>Dynabeads Protein G</td><td>Novex, Thermo Fisher Scientific</td><td>10003D</td><td></td></tr><tr><td>DRBP76 (NF90/NF110) antibody</td><td>BD Transduction Laboratories</td><td>612154</td><td>use 1:1,000 for WB and 1:100 for ICC/PLA</td></tr><tr><td>RBM3 antibody</td><td>ProteinTech</td><td>14363-1-AP</td><td>use 1:1,000 for WB and 1:100 for ICC/PLA</td></tr><tr><td>Lamin A/C antibody</td><td>Cell Signaling Technology</td><td>#2032</td><td>use 1:1,000 for WB&amp;nbsp;</td></tr><tr><td>anti-GAPDH antibody</td><td>Abcam</td><td>ab8245</td><td>use 1:1,000 for WB&amp;nbsp;</td></tr><tr><td>normal rabbit IgG</td><td>Santa Cruz</td><td>sc-2027</td><td></td></tr><tr><td>anti-rabbit IgG, HRP-linked secondary antibody</td><td>Cell Signaling Technology</td><td>#7074</td><td>use 1:5,000 for WB&amp;nbsp;</td></tr><tr><td>anti-mouse HRP secondary antibody</td><td>Carl Roth</td><td>4759.1</td><td>use 1:5,000 for WB&amp;nbsp;</td></tr><tr><td>Clarity Western ECL Blotting Substrate</td><td>Bio-Rad</td><td>#1705060</td><td></td></tr><tr><td>NuPAGE Novex 4-12% Bis-Tris Gel</td><td>Novex, Thermo Fisher Scientific</td><td>NP0321BOX</td><td></td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP0002</td><td></td></tr><tr><td>NuPAGE Transfer Buffer (20x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP00061</td><td></td></tr><tr><td>Amersham Hypond P 0.2 PVDF membrane</td><td>GE Healthcare Life Sciences</td><td>10600021</td><td></td></tr><tr><td>Poly-D-Lysine 8 Well Culture Slide</td><td>Corning BioCoat</td><td>354632</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Gibco, Thermo Fisher Scientific</td><td>PCN5000</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L Antibody), Alexa Fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11001</td><td></td></tr><tr><td>Goat anti-rabbit IgG (H+L Antibody), Alexa Fluor 568 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11011</td><td></td></tr><tr><td>4&amp;prime;, 6-Diamidin-2-phenylindol (DAPI)</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Duolink PLA probe Anti-mouse PLUS</td><td>Sigma</td><td>DUO92001</td><td></td></tr><tr><td>Duolink PLA&amp;nbsp; probe Anti-rabbit MINUS</td><td>Sigma</td><td>DUO92005</td><td></td></tr><tr><td>Duolink Detection Reagents Red</td><td>Sigma</td><td>DUO92008</td><td></td></tr><tr><td>Duolink Wash Buffers Fluorescence</td><td>Sigma</td><td>DUO82049</td><td></td></tr><tr><td>Duolink Mounting Medium with DAPI</td><td>Sigma</td><td>DUO82040</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma</td><td>81381</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>AX-70</td><td></td></tr><tr><td>CCD camera</td><td>SPOT</td><td>Insight 2MP Firewire</td><td></td></tr><tr><td>X-ray film</td><td>Fujifilm</td><td>Super RX</td><td></td></tr><tr><td>Film processing machine</td><td>Fujifilm</td><td>FPM-100A</td><td></td></tr></tbody></table>

visualization of protein-protein interaction in nuclear and cytoplasmic fractions by co-immunoprecipitation and in situ proximity ligation assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Şekil 1, NF90 ve RBM3 hem çekirdek proteinleridir ve sadece küçük bir fraksiyonu sitoplazmada mevcut olduğunu ortaya koymaktadır. Özellikle, RBM3 için pozitif boyandı üç farklı bant vardır. sadece 20 kDa altında küçük RBM3 doğru boyutunu yansıtır (RBM3 tahmin molekül ağırlığı 17 kDa olan). iki bant kökeni araştırılması devam etmektedir. yem protein olarak RBM3 ile ko-immünopresipitasyon deneyleri NF90-RBM3 etkileşimleri çekirdek ve sito...

Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Co-immunoprecipitation tarafından Nükleer ve Sitoplazmik Kesirler Protein-protein Etkileşim görselleştirme ve<em&gt; In Situ</em&gt; Proximity ligasyon Testi

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

visualization-protein-protein-interaction-nuclear-cytoplasmic

Research

JoVE Journal

Biochemistry

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

12.7K Görüntüleme.  University of Basel. Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Video: Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

Proximity Ligation Testi ile Süspansiyon Hücre Kültürlerinde DNA Hasarına Bağlı Protein Komplekslerinin Saptanması ve Görselleştirilmesi

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biyokimya

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Heterodimerization in Situ yakınlık ligasyonu tahlil kullanarak Protein izoformlarının tespiti

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Multipleksed Co-İmmünopreyağış Kullanarak Protein Etkileşim Ağı Dinamiğinin Ölçülmesi

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Büyük Ölçekli Nükleer Etkileşimom Profilleme için Etiketsiz İmmünoprekütle Spektrometresi İş Akışı

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

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

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Algılama<em&gt; In Situ</emEn&gt; Protein-protein kompleksleri<em&gt; Drosophila</emProximity ligasyon Testi Kullanımı&gt; Larva Nöromüsküler Junction

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Nörobilim

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Görüntüleme Protein-protein etkileşimleri In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Biyoloji

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Bakteri, Maya ve İnsan Hücrelerinde Geçici Olarak Oluşan Moleküler Şaperon Meclislerinin Yerinde İzlenmesinde

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

ER-Mitokondri Temas Bölgelerinde Endojen Organel İçi Protein Etkileşimlerinin Yakınlık Ligasyon Testleri ile Görselleştirilmesi ve Miktarının Belirlenmesi

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

<meta charset="utf8"></head><body>DNA hasarında protein etkileşimi ve lokalizasyonunun değerlendirilmesi

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

<meta charset="utf8"></head><body>Yazar Spotlight: DNA Hasar Yanıtındaki Protein Etkileşimlerini Karakterize Etmek için Yakınlık Ligand Testini Gama-H2AX Boyama ile Birleştirme

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

DNA Hasar Bölgelerindeki Proteinleri Lokalize Etmek için Yakınlık Ligand Testi

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

Co-immunoprecipitation tarafından Nükleer ve Sitoplazmik Kesirler Protein-protein Etkileşim görselleştirme ve

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