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.免疫共沉淀<ol><li>种子HEK293细胞以每孔2×10 5个细胞在一6孔板的2ml Dulbecco氏改良的Eagle氏培养基（DMEM）中的补充有10％胎牛血清（FBS）和100U / mL的青霉素-链霉素（青霉素-链霉素） 。 </li><li>生长细胞48小时，在37℃，5％的CO 2。 </li><li>洗涤细胞用冷的磷酸盐缓冲盐水（PBS）的三倍。收获细胞，在500×g离心5分钟，离心在4℃下。 </li><li>使用商业细胞核和细胞质提取试剂准备细胞核和细胞质分数。按照制造商的一些修改指令。 <ol><li>用3×10 6细胞对一种共免疫沉淀实验（来自3个孔一个6孔板的约90％汇合）。加入300微升冷的细胞质提取液（CER I），涡旋15秒的最高速度。 </li><li>孵育在冰上30分钟。孵化期间，vortex对于5秒，每10分钟。 </li><li>添加16.5微升冷的细胞质萃取液II（CER II），涡旋5秒，并培育冰上5分钟。 </li><li>涡流在4℃下5秒并离心16,000×g离心5分钟。 </li><li>转移上清液（胞浆提取物）到一个新的预冷的管，并保持在冰上直到使用。 </li><li>通过每次上下吹打五次用1ml冷的PBS洗涤不溶性颗粒（含核）三次。后洗净除去PBS。 </li><li>加入150微升冷的核提取液（NER），涡旋15秒，冰上孵育1小时。在孵化，涡旋15秒和吸管10次用200微升末端每隔10分钟。 </li><li>涡旋15秒并离心16,000 xg离心在4℃下10分钟。 </li><li>转移上清液（核抽提物）到一个新的预冷的管，保持在冰上直到使用。 </li></ol></li><li>取出核和细胞质提取物的体积的10％的每个作为输入。 </li><li>一个DD冷PBS到剩余提取物1毫升的最终体积。置于冰上直至使用。 注:使用蛋白G偶联的磁珠时预结算不是必需的。然而，如果背景是高，通过温育将40μl蛋白G偶联的磁珠（50％浆液）和1ml这个步骤在4℃下在旋转器上30分钟稀释溶胞产物进行预清除。从与磁性架珠粒分离上清液（预澄清的溶胞产物）。丢弃的珠子。 </li><li>与蛋白G偶联的磁珠夫妇初级抗体，孵育40微升蛋白G偶联的磁珠（50％浆液）与4微克兔多克隆抗RBM3抗体或在200μlPBS中兔IgG（阴性对照）加Tween 20缓冲液（PBST，0.02％吐温20）在室温（RT）下在旋转器上40分钟。 </li><li>单独的抗体 - 偶联珠和上清液用磁力架，并弃去上清液。用200μlPBST（0.02％）洗珠一次。 </li><li>加1毫升稀释裂解物（或者如果需要的话，预澄清的溶胞产物）的珠，并在4℃下在旋转器上孵育过夜。 </li><li>在第二天，用磁力架分离珠和上清液，并弃去上清液。在4℃下在旋转器上洗3×10分钟用0.5ml PBST（0.02％）为每个管20 rpm的固定速度。 </li><li>通过加入40微升样品缓冲液（1×商业样品缓冲液和50mM 1,4-二硫苏糖醇（DTT））洗脱从珠蛋白。热，在70℃下进行10分钟。降速和传输液体到新的1.5 ml管。 </li><li>稀释样品缓冲输入裂解物（最终浓度:1×商业样品缓冲液和50mM DTT）和加热在70℃下进行10分钟。从最后一步到一个预制4-12％的Bis-Tris凝胶负载输入和免疫沉淀的蛋白质。每口井的装载量不宜超过20微升。在1×商业运行缓冲液进行电泳在冰上35分钟。 注:加载细胞核和细胞质提取物S IN 1:2的比例（V / V），这反映了细胞的相同的初始量。 </li><li>转移蛋白至PVDF膜在1×商业转印缓冲液中在30伏2小时，在4℃。 </li><li>用5％的块膜在RT脱脂在PBST乳（0.1％吐温20），用于在振荡器上40分钟。 </li><li>孵育初级抗体膜（在1都稀释:1,000）在PBST（0.1％），在4℃下过夜。 </li><li>在RT在摇床上洗涤3×10分钟用PBST（0.1％）。 </li><li>孵育辣根过氧化物酶（HRP）缀合的膜的抗小鼠或抗兔次级抗体（在1都稀释:5,000）在PBST（0.1％）在RT 1小时。 </li><li>在RT在摇床上洗涤3×10分钟用PBST（0.1％）。 </li><li>使用0.2毫升增强的化学发光（ECL）基板每平方厘米膜混合物，孵育在室温下5分钟。避免这一步，除了起红灯全亮。 </li><li>丢弃液体并暴露于X射线胶片的胶片盒。使用自动薄膜PR开发薄膜ocessing机。 </li></ol> 2.免疫细胞化学和接近结扎分析<ol><li>种子补充有10％FBS和100U / ml的青霉素-链霉素的HEK293细胞以每室1.5×10 4个细胞在一个8腔聚D-赖氨酸涂覆的滑动0.4毫升DMEM中。 </li><li>生长细胞48小时，在37℃，5％的CO 2。 </li><li>抽吸培养基，并修复用4％多聚甲醛（PFA）在室温10分钟的细胞。 </li><li>抽吸PFA，并与每室0.5毫升的PBS洗涤3×10分钟。 </li><li>通透和嵌段细胞用在PBS中的0.5％的Triton X-100和5％正常山羊血清（NGS）在RT 1小时。 </li><li>在4℃下在0.1％的Triton X-100和5％NGS的PBS中的一抗在振荡器上孵育过夜。稀释的小鼠单克隆抗NF90抗体和兔多克隆抗RBM3抗体1:在PBS 100为双重染色。为阴性对照，独立地省略或两个初级抗体，而在第三控制省略两种抗体。 注:请不要使用商业阻塞和稀释试剂。 </li><li>洗3×10分钟用0.5毫升PBS中每室。 </li><li> 受免疫或接近连接试验协议 <ol><li>免疫细胞化学<ol><li>用1孵育:500稀释绿色荧光染料 - 偶联的抗 - 小鼠和红色荧光染料偶联的抗 - 兔二级抗体在RT 1小时。从这一步起，避免了光。 </li><li>在PBS 5000在RT 10分钟:用4&#39;，6- diamidin -2-苯基吲哚（DAPI）对细胞核稀释1。 </li><li>洗3×10分钟用0.5毫升PBS中每室。 </li><li>移除载玻片和干燥腔室。 250μ安装，安装每张幻灯片中。 </li></ol></li><li>接近结扎法<ol><li>准备接近结扎分析探针。混合和稀释附着有不同的寡核苷酸，可以结扎日两个接近连接测定探针（抗小鼠和抗兔二级抗体粗加在结扎溶液中的其它两个寡核苷酸）两者1:5的0.1％的Triton X-100和5％NGS的PBS，用320微升的总体积为一个8腔室滑动件（ 每平方厘米约40微升） 。孵育在室温20分钟。 </li><li>从载玻片移除腔室，并添加稀释探针。孵育在湿度培养箱中于37℃进行1小时。 </li><li>准备连接解决方​​案。混合8微升连接酶，64微升5×连接股票和248微升H 2 O. </li><li>分接从滑动的液体，并在1×洗涤缓冲液洗涤2×5分钟（由试剂盒提供）。 </li><li>添加连接溶液，并在37℃的湿度培养箱温育30分钟。 </li><li>准备扩增溶液。混合4微升聚合酶，64微升5倍放大的股票和252微升H 2 O.从这一步起，避光。 </li><li>从滑动轻按脱液，并在1×洗涤缓冲液A洗涤2×2分钟</li><li>添加耳放lification溶液，并在黑暗湿度培养箱孵育在37℃下为100分钟。 </li><li>分接从滑动的液体，并在1×洗涤缓冲液B洗2×10分钟（由试剂盒提供）。洗在0.01X洗涤液B. 1分钟</li><li>擦干幻灯片，每张幻灯片250微升商业安装介质（用DAPI）的安装。 </li></ol></li></ol></li><li>检查用10X目镜透镜和20X物镜的显微镜下的荧光信号。通过CCD照相机获得的图像。 </li></ol>

<ol>
	<li>Pati&#241;o, C., Haenni, A. L., Urcuqui-Inchima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF90+isoforms,+a+new+family+of+cellular+proteins+involved+in+viral+replication?.">NF90 isoforms, a new family of cellular proteins involved in viral replication?.</a> Biochimie. 108, 20-24 (2015).</li><li>Shim, J., Lim, H., R Yates, J., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+export+of+NF90+is+required+for+interleukin-2+mRNA+stabilization.">Nuclear export of NF90 is required for interleukin-2 mRNA stabilization.</a> Mol Cell. 10 (6), 1331-1344 (2002).</li><li>Sakamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NF90-NF45+complex+functions+as+a+negative+regulator+in+the+microRNA+processing+pathway.">The NF90-NF45 complex functions as a negative regulator in the microRNA processing pathway.</a> Mol Cell Biol. 29 (13), 3754-3769 (2009).</li><li>Dresios, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+stress-induced+protein+Rbm3+binds+60S+ribosomal+subunits,+alters+microRNA+levels,+and+enhances+global+protein+synthesis.">Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.</a> Proc Natl Acad Sci. 102 (6), 1865-1870 (2005).</li><li>Danno, S., Itoh, K., Matsuda, T., Fujita, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+expression+of+mouse+Rbm3,+a+cold-shock+protein,+in+Sertoli+cells+of+cryptorchid+testis.">Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of cryptorchid testis.</a> Am J Pathol. 156 (5), 1685-1692 (2000).</li><li>Wellmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-regulated+expression+of+the+RNA-binding+proteins+RBM3+and+CIRP+by+a+HIF-1-independent+mechanism.">Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism.</a> J Cell Sci. 117 (Pt 9), 1785-1794 (2004).</li><li>Zhu, X., Zelmer, A., Kapfhammer, J. P., Wellmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-inducible+RBM3+inhibits+PERK+phosphorylation+through+cooperation+with+NF90+to+protect+cells+from+endoplasmic+reticulum+stress.">Cold-inducible RBM3 inhibits PERK phosphorylation through cooperation with NF90 to protect cells from endoplasmic reticulum stress.</a> FASEB J. 30 (2), 624-634 (2016).</li><li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Verhelst, J., De Vlieger, D., Saelens, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-immunoprecipitation+of+the+Mouse+Mx1+Protein+with+the+Influenza+A+Virus+Nucleoprotein.">Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.</a> J Vis Exp. (98), (2015).</li><li>S&#246;derberg, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation.">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nat Methods. 3 (12), 995-1000 (2007).</li><li>Jarvius, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+phosphorylated+platelet-derived+growth+factor+receptor+beta+using+a+generalized+proximity+ligation+method.">In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method.</a> Mol Cell Proteomics. 6 (9), 1500-1509 (2007).</li><li>Liu, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein-protein+interactions+in+cross-talk+pathways+reveals+CRKL+protein+as+a+novel+prognostic+marker+in+hepatocellular+carcinoma.">Analysis of protein-protein interactions in cross-talk pathways reveals CRKL protein as a novel prognostic marker in hepatocellular carcinoma.</a> Mol Cell Proteomics. 12 (5), 1335-1349 (2013).</li></ol>

The authors have nothing to disclose.

有几个好处，以及这两种方法的缺点。作为一个相对较新的技术中，邻近连接测定的一个明显的优点是阐明在单细胞水平，而不是一个批次异质细胞蛋白质 - 蛋白质相互作用的可行性。具有较高的幅度和分辨率（ 例如 ，通过共聚焦显微镜）图像提供了通过计算单荧光斑点进行量化的可能性。相反，用Western印迹免疫共沉淀技术的常规组合只能半定量蛋白质条带，这主要是因为一个合适的负载控制是难以确定的IP样品。此外，当蛋白质-蛋白质相互作用是弱或短暂，大量的生物材料， 例如细胞或组织，需要共免疫沉淀或与熔融标签的人工过度表达系统的应用，以提高检测的相互作用的机会。另外，检测Ť具有高灵敏度echniques如质谱可以提高实验质量。但是，在相对于起始物质的量的邻近连接测定方法具有明显的优势。只有少数细胞是必需的，提供的抗体的高品质中给出。另外，组织样品中， 在不同的组织结构和细胞类型的蛋白质-蛋白质相互作用的原位可视可通过邻近连接测定实现的，在类似的模式为正常免疫组织化学。相比之下，共免疫沉淀是通过其性质不适于显示蛋白质 - 蛋白质相互作用的空间分布。 在大核小，但细胞质车厢细胞，接近结扎测定方法只限于在分析核质分布和传统的免疫共沉淀有其自身的优势。例如，在T淋巴细胞的细胞系， 例如 Jurkat细胞，核细胞质DISTRI通过邻近连接测定技术NF90-RBM3相互作用butions受到限制，因为核占据在细胞内几乎整个空间，并且难以识别细胞质隔室的边界。这可以是在一般的免疫细胞化学一个共同的问题，当核 - 细胞质比率极不平衡。然而，共免疫沉淀在这种情况下，不受此限制。 关于邻近连接测定一个特殊的问题是来自接近连接测定的信号是否表示直接或间接的蛋白质 - 蛋白质相互作用。无论NF90和RBM3具有RNA结合特性及其相互作用是依赖于RNA，在我们以前的研究7日报道。因此，用RNase细胞裂解物的预处理溶解NF90和RBM3并没有什么之间的相互作用是通过共免疫沉淀法7检测的。然而，接近连接测定信号不受影响即使RNA依赖性蛋白质 - 蛋白质相互作用失去RNA种类，因为当两个蛋白质之间的距离小于40纳米，这通常被认为是作为一个直接相互作用它产生。邻近连接测定的这种性质可以克服从共免疫沉淀的技术问题，如核糖核酸酶释放在细胞裂解物中，可能取消要求的RNA调解的相互作用。然而，在另一方面，邻近连接测定方法还可以产生假阳性信号，如果RNA依赖性蛋白质 - 蛋白质相互作用实际上并不由于缺乏的在生理条件下特异性RNA存在。 值得特别注意的另一个问题是初级抗体的特异性和蛋白同种型，前体和蛋白聚集的可能性。我们观察到免疫印迹，长期NF90亚型，NF110，在这两个细胞核和细胞质在HEK293细胞更丰富相比，NF90（FI古尔1）。然而，免疫共沉淀解开NF110的较高结合亲和力RBM3特别是在细胞核中。三个主要带中RBM3印迹发现在细胞核中，但只有20 kDa的正常RBM3带在细胞质主要观察到。是否另外两个50 kDa和100 kDa的条带反映RBM3同种型，前体，具有结合的RBM3蛋白质聚集体或是由于非特异性背景仍有待阐明。相反，由于邻近连接测定是基于免疫染色，邻近连接测定不能区分之间不同的蛋白质同种型，前体，聚集体或非特异性染色的差异，而共免疫沉淀可以提供抗体特异性或蛋白同种型和前体比的详细信息邻近连接测定调查蛋白质 - 蛋白质相互作用。关于RBM3，考虑到核观察到的所有三个RBM3波段时，印迹结果是一个主要的核一致localizati在RBM3由免疫观察。 总之，这两种免疫共沉淀和邻近连接测定技术具有内在的优点和限制。在许多情况下，它们产生一致的结果，但它是有益的是使用这两种技术，当系统地研究某些蛋白质 - 蛋白质相互作用。然而，在特殊情况下，或具有特定的目的，人们可以显示优势比其他。最近，接近连接测定已用于屏幕蛋白质-蛋白质相互作用，以开发新的预后标志物12。蛋白 - 蛋白相互作用被认为是更可靠的生物标记物比单个蛋白质。在未来，邻近连接测定可潜在成为分析蛋白质 - 蛋白质相互作用中诊所的诊断和预后标志物快速和可靠的方法，虽然这些生物标志物的鉴定和表征仍然需要传统的共immuoprecipication箱的ThOD。

核因子90（NF90）是一个多同种型蛋白质与许多功能，包括白细胞介素-2的后转录和miRNA生物发生1-3的调节响应于病毒感染，调节。 RBM3是一种RNA结合蛋白，参与翻译和miRNA生物发生，并且可以通过各种紧张，包括低温和缺氧4-6诱导。最近，我们发现NF90和RBM3的蛋白复合物7。 NF90和RBM3的相互作用是必不可少的调节在折叠蛋白反应7蛋白激酶RNA样内质网激酶（PERK）活性。无论NF90和RBM3主要位于细胞核中，但NF90的一小部分，并RBM3航天飞机进入细胞质并结合有彼此为特定的功能， 如调节PERK活动。因此，可视化NF90-RBM3相互作用的分布在亚细胞区室，其可以指示是很重要它们在各自的舱室各种角色。 几十年前，酵母双杂交（Y2H）的开发，以检测两种蛋白质8之间的相互作用。然而，由于融合蛋白的人工建造，假阳性结果限制了这种方法的应用。很长一段时间，免疫共沉淀是分析蛋白质-蛋白质相互作用，特别是在内源性条件9的主要技术。为了分析共同免疫沉淀的蛋白质复合物，蛋白质印迹是最方便的技术，而当超灵敏性和精确性期望使用质谱法。近年来，接近连接测定已发展为一种新的方法来检测在原位 10,11在两个细胞和组织中蛋白-蛋白相互作用。 在这里，我们比较捕捉NF9菌株最流行的免疫共沉淀法和相对新颖的接近结扎测定方法在亚细胞组分0 RBM3互动。我们还讨论的优点和两种技术的局限性。

<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.

图1表明，NF90和RBM3都是核蛋白和只有一小部分是存在于细胞质中。值得注意的是，存在用于RBM3染色阳性三个不同的频带。最小的正下方的20kDa反映RBM3的正确大小（RBM3的预测分子量为17 kDa的）。其他两个波段的起源还有待调查。与RBM3作为诱饵蛋白共免疫沉淀实验表明NF90-RBM3相互作用是主要存在于核和细胞质少数。免疫共沉淀的数据支持每个单独的蛋白质的本地化。 如图2A所示，NF90和RBM3主要位于细胞核而且在原位细胞质中。这两种蛋白质表现出在两个隔室完美的共定位。接近结扎测定显示模式的NF90-RBM3相互作用，这是非常类似于传统免疫细胞化学，在大多数细胞中细胞核大多数相互作用。只有细胞的一小部分证明NF90-RBM3相互作用的主要细胞质分布。 加在一起，共免疫沉淀和邻近连接测定技术反映基本上在核和细胞质区室的蛋白质 - 蛋白质相互作用的相同的分布图案。 <img alt="图1" src="/files/ftp_upload/55218/55218fig1.jpg" /> 图1:NF90和RBM3及其在HEK293细胞的核质馏分相互作用的免疫印迹分析。核和细胞质提取物装载到SDS-PAGE凝胶中的一个比为1:2（V / V），反映细胞的相同量的在提取协议说明。拉明和GAPDH用作核和细胞质的标记，分别为。免疫共沉淀，用抗RBM3抗体或兔IgG作为阴性对照。核和细胞质提取物还用抗RBM3抗体孵育在1:2的比例（V / V）分别。在NF90印迹上带指示110 kDa的长同种型NF110。 N:核提取物; C:细胞质提取物，IP:免疫沉淀。蛋白质标记物标记为RBM3阳性条带。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55218/55218fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55218/55218fig2.jpg" /> 图2:免疫组化和HEK293细胞接近结扎分析。 （A）的HEK293细胞与抗NF90（绿色）和抗RBM3（红色）的抗体的双染色。箭头显示细胞NF9菌株明确细胞质共定位0和RBM3。细胞核用DAPI（蓝色）复染色。 （B）中邻近连接测定用抗NF90和在HEK293细胞中的抗RBM3抗体。红色荧光斑点表明NF90-RBM3相互作用。箭头显示在细胞质NF90-RBM3相互作用。细胞核用DAPI（蓝色）复染色。阴性对照1（NC 1）:初级抗体省略。只有RBM3单一的初级抗体:阴性对照2（NC 2）。阴性对照3（NC 3）:NF90单个主只有抗体。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55218/55218fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

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

Nanyang Technological University

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 次观看.  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.

视频: 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.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

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

科研

生物化学

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.

原位近接结扎法检测蛋白异构体的 Heterodimerization

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.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

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.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

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.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

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

发育生物学

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.

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

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

神经科学

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.

成像蛋白质 - 蛋白质相互作用在体内</em

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

生物学

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.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

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.

通过邻近连接测定可视化和定量ER-线粒体接触部位的内源性细胞器内蛋白相互作用

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>评估 DNA 损伤中的蛋白质相互作用和定位

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>作者聚焦：将邻位配体检测与 γ-H2AX 染色相结合以表征 DNA 损伤反应中的蛋白质相互作用

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

邻位配体检测，用于定位 DNA 损伤位点的蛋白质

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

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和

摘要

探索更多视频

此视频中的章节