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>

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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?...

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 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.

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?...

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

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

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.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

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

科研

生物化学

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 ...

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.

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

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.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

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.

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 ...

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.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

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.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

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.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

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.

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 ...

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.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

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

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和

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