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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td>High glucose 
			4500 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 1:1000 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 1:1000 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 1:1000 for WB&#160;</td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Abcam</td>
			<td>ab8245</td>
			<td>use 1:1000 for WB&#160;</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-lined secodary antiboy</td>
			<td>Cell Signaling Technology</td>
			<td>#7074</td>
			<td>use 1:5000 for WB&#160;</td>
		</tr>
		<tr>
			<td>anti-mouse HRP secondary antibody</td>
			<td>Carl Roth</td>
			<td>4759.1</td>
			<td>use 1:5000 for WB&#160;</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>1,4-Dithiothreitol (DTT)</td>
			<td>CarlRoth</td>
			<td>6908.1</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>Super RX X-ray film</td>
			<td>Fujifilm</td>
			<td>4741029230</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&#8242;, 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&#160; 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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Yöntem HCN1 ve TRIP8b arasında protein-protein etkileşimi küçük molekül önleyicilerinin tespit edilmesi için

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Biyokimya

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 ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hidrolizat tavuk tüyleri ve kozmetik kendi uygulamasında hazırlanması

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

X-ışını kristalografisi ve biyofiziksel teknikleri immünglobulin katlanmış glikoproteinlerin karakterizasyonu

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

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 ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Protein katlanması, taşıma ve canlı hücreler radyoaktif darbe Chase tarafından bozulması Analizi

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

Transkripsiyon protein-protein etkileşimlerini Incelemek için Biolayer Interferometri (BLı) uygulaması

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

Amiloid Fibrils ile Beyin Mitokondrisi ile Etkileşimler ve Membran Permeabilizasyonu

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

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 ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Membran Protein-Protein Etkileşim Analizi için Yerli Hücreli Membran Nanopartiküller Sistemi

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...