Guikema实验室的研究由荷兰科学研究组织（VIDI授权016126355）和"Stichting Kinderen Kankervrij"KiKA（项目252）的创新研究奖励计划资助。

1.细胞治疗和DNA损伤诱导<ol><li>在补充有20％FCS，50μMβ-巯基乙醇，2mM L-谷氨酰胺，100U / mL青霉素和100μg/ mL链霉素的IMDM中培养人BCR-ABL + B细胞急性成淋巴细胞细胞系BV173或SUP-B15在5％CO 2的气氛中37℃。在6孔板中以5mL /孔计数细胞并以2×10 6细胞/ mL平板。 注意:任选地，可以使用各种来源的悬浮细胞系。 </li><li> 为了阻止细胞周期G1期的BCR-ABL + B-ALL细胞，加入5μM的伊马替尼甲磺酸盐（STI571），并孵育过夜。 <ol><li>任选地，当研究整个细胞周期或BCR-ABL阴性细胞类型中的蛋白质 - 蛋白质相互作用时，省略此步骤。 </li></ol></li><li> 通过向培养物中加入50ng / mL新卡那霉素（NCS）诱导DNA损伤，并孵育2小时。 <ol><li>或者，通过使用放射源[ 137 Cs]以0.5Gy / min以5Gy的剂量照射细胞来诱导DNA损伤。照射后，让细胞在37℃，5％CO 2气氛中恢复2小时。 </li></ol></li><li>任选地，为了评估ATM激酶活性对DNA损伤的反应，在加入NCS或照射治疗之前加入5μM的ATM激酶抑制剂KU55933。 </li><li>通过在烧杯或锥形烧瓶中的50mL 1x PBS的顶部层叠5g级分的V BSA，制备1×PBS + 10％BSA，并放置在室温下直到BSA溶解。不要摇晃或搅拌，因为会引起大量的起泡。缓慢过滤0.22μm过滤器并保持在冰上。 </li><li>收获细胞，用冰冷的1x PBS洗涤两次。计数细胞并在1×PBS + 10％BSA中以2×10 6 / mL重悬。保持细胞在冰上。 </li></ol>细胞离心，固定和渗透<ol><li>准备4％多聚甲醛溶液1倍PBS新鲜。加入2 g PFA至50 mL 1x PBS，并在55°C振荡水浴中孵育，直至溶液澄清。通过0.22μm过滤器过滤溶液，并在RT存储下使用。 注意:任选地，4％PFA固定剂可以在-20°C下冷冻保存。冻结后，解冻一次即可使用。否则，PBS中的4％PFA溶液可以直接购买。 </li><li>仔细研磨76毫米×26毫米的显微镜幻灯片与无绒组织，并通过将其放置在Coplin罐中的96％乙醇中5分钟来脱脂。让显微镜空气干燥10分钟。 </li><li>将显微镜载玻片放入面积为6 mm的细胞离心/细胞学漏斗中。通过将50μL1×PBS + 10％BSA移液到每个漏斗中预先涂覆载玻片，并以500rpm离心1分钟。 </li><li>向每个漏斗加入100μL细胞悬浮液（等于0.2×10 6个细胞），并以500rpm离心5分钟。对于每种抗体组合，需要ast 4制剂（双抗体实验;两个单抗体对照;无抗体对照）。 </li><li> 小心地拆卸漏斗，并确保不要触摸点。立即进行样品固定。 <ol><li>任选地，将载玻片风干至少30分钟。风干后，将薄片单独包裹在铝箔中，储存于-20°C直至使用。当使用冷冻制剂时，请确保解冻用纸巾包裹的载玻片，以防止幻灯片上露出冷凝。 </li></ol></li><li>使用PAP笔式液体阻塞剂（5mm尖端）在斑点周围绘制一个圆圈（大约直径15-20 mm）。 </li><li>向每个斑点加入50μL的4％PFA固定溶液，并在室温下孵育30分钟。 </li><li>在轻轻搅拌下，在室温下，在Coplin罐中用1x PBS洗涤细胞3次5分钟。 </li><li>使用无绒毛组织将斑块周围的斑块干燥，并使用pI从尽可能多的液体中除去尽可能多的液体宠物没有触摸现场，或通过倾斜幻灯片。向每个斑点加入50μL的0.25％Triton-X在1×PBS中，并在室温下不搅拌孵育10分钟。 </li><li>在轻轻搅拌下，在Coplin罐中，在TBS（TBS-T）中的约50-60mL的0.05-60ml的Tween-20中洗涤载玻片3次5分钟。可以以这种方式同时处理10个幻灯片。 </li></ol> 3.阻断和一级抗体孵育<ol><li>点击TBS-T，用无棉绒纸巾围绕斑点干燥。使用移液管尽可能多地从现场清除液体，而不必接触该位置，或通过倾斜滑块。短暂旋转阻塞溶液，并向每个点加入一滴（约40μL）。在预热的加湿室中在37℃下孵育1小时。 </li><li>在商业抗体稀释剂，涡旋抗体稀释剂使用前，以1:100混合山羊抗ATM和1:100的兔抗磷酸化Ser15-p53，制备抗体混合物。用于控制实验制备仅含有山羊抗ATM和仅抗兔抗磷酸化Ser15-p53抗体（单抗体对照）的抗体稀释液。 </li><li>点击阻塞溶液，但注意不要让细胞在加入抗体稀释液之前干燥。加入30μL每种抗体混合液稀释液，并在4℃下在加湿室中孵育过夜。 </li><li> 通过将一袋缓冲液A和缓冲液B混合物的内容物溶解在最终体积为1000mL的水中来原位洗涤缓冲液A和洗涤缓冲液B。 <ol><li>或者，通过将8.8g NaCl，1.2g Tris-碱和0.5mL Tween-20溶解在800mL水中来制备洗涤缓冲液A。使用HCl将pH调节至7.4，加入最终体积为1000 mL。 </li><li>或者，通过将5.84g NaCl，4.24 Tris-碱和26.0g Tris-HCl溶于500mL水中制备洗涤缓冲液B.使用HCl将pH调节至7.5。加入水至最终体积为1000 mL。 </li><li>过滤两种溶液通过0.22μm过滤器。 </li></ol></li><li>用轻轻搅拌的室温下，在Coplin罐中用1x 原位洗涤缓冲液A洗涤载玻片2次5分钟。 </li></ol> 4. PLA探针，连接和扩增<ol><li>通过在商业抗体稀释液中稀释1:5的抗山羊PLUS和抗兔MINUS来制备PLA探针。从幻灯片中抽出1x 原位洗涤缓冲液A，并向每个斑点加入30μLPLA探针混合物。在预热的加湿室中37℃孵育1小时。 注意:只要所应用的一抗在不同的物种中升高，PLA二级抗体PLUS和MINUS PLA探针的组合也可以使用（抗小鼠，抗兔，抗山羊）包含一个PLUS探针和一个MINUS探针。 </li><li>在轻轻摇动的同时，在Coplin罐中用〜50-60mL 1x 原位洗涤缓冲液A洗涤载玻片2次5分钟。/ LI&gt; <li>通过向39μL的连接溶液中加入1μL的连接酶，在冰上准备连接连接酶溶液，并向每个点加入40μL的连接连接酶溶液。在预热的加湿室中在37℃下孵育30分钟。 </li><li>在轻轻搅拌下，在Coplin罐中用〜50-60mL 1x 原位洗涤缓冲液A洗涤载玻片2次5分钟。 </li><li>将放大聚合酶混合物在冰上通过在水中1:5稀释扩增储备溶液来制备。然后向每个斑点的39.5μL的1x扩增溶液中加入0.5μL的聚合酶。 注意:扩增试剂是光敏感的，应防止直接照射。扩增试剂有四种不同的颜色（RED:激发594nm，发射624nm; FarRED:激发644nm，发射669nm; ORANGE:激发554nm，发射576nm; GREEN:激发501nm，发射523nm）确保显微镜设置可用适当的激发特性和滤镜来对这些颜色进行成像。 </li><li>点击1x 原位洗涤缓冲液A，每个点加入40μL的Amplification-Polymerase混合物。在预热的加湿室中在37℃下孵育100分钟。 </li></ol> 5.安装和成像<ol><li>通过向99 mL水中加入1 mL 1x洗涤缓冲液B来制备0.01x 原位洗涤缓冲液B。 </li><li>点击放大聚合酶混合物，并用轻轻搅拌的RT在Coplin罐中用1x 原位洗涤缓冲液B洗涤载玻片2次10分钟。 </li><li>在0.01x洗涤缓冲液B中洗涤载玻片1分钟</li><li>分离多余的0.01x洗涤缓冲液B，并用无绒纸巾将斑块周围的玻片干燥。使用移液管尽可能多地从现场清除液体，而不必接触该位置，或通过倾斜滑块。 </li><li>在不含DAPI的安装介质中稀释含DAPI的安装介质1:5，以避免核的过度染色。 </li><li>将25-30μL含有DAPI的1:5 DAPI安装介质添加到24 mm x 24 mm的盖玻片上，并用滑块拾起盖板，施加轻微的压力，以确保没有被困的气泡。用指甲油密封盖玻片，并在成像前等待至少15分钟。 </li><li> 使用（共焦）荧光显微镜分析载玻片。 <ol><li>在至少4个获取的图像场中计数每个细胞的点状PLA信号的数目（20x目标，基于使用处理的细胞中7±5个信号的预期平均值的功率计算，每个条件或抗体组合计数至少20个细胞，和未经处理的细胞中的2±2个信号，I型误差的概率为0.05，功率为80％，如前所述15 ）。 注意:最佳分辨率是通过使用软件包（ 例如 ，ImageJ，Leica Acquisition Suite（LAS））获取Z-stack和去卷积来实现的。信号在DAPI染色定义的核周期内，ls应该很容易识别。 </li><li>不要计数出现在细胞核外的信号。通常，NCS处理或γ照射导致每个细胞约5-10个phosho-p53 / ATM PLA信号。 "单抗体"对照可能会产生一些背景信号，但每20个细胞中每1个不得超过1-2个PLA信号。 </li><li>出版和演示目的，使用40X或63X浸油目标拍摄图像。幻灯片可以在4°C储存，并应防止曝光。 </li></ol></li></ol>

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作者没有什么可以披露的。

在本报告中，证明PLA可用于确定和可视化悬浮细胞培养物中蛋白质之间的特异性相互作用。值得注意的是，这里描述的方案不限于DNA修复复合物的研究，而且还适用于可视化和定量悬浮细胞培养物中的其他蛋白质 - 蛋白质相互作用。显示当暴露于DNA损伤诱导剂时，ATM激酶与G1-阻滞的BCR-ABL + B-ALL细胞中的磷酸化p53相互作用。以前，我们使用PLA技术来表明ATM和FOXO1转录因子在BCR-ABL + B-ALL细胞中相互作?...

DNA损伤触发高度受限制的一系列事件，涉及蛋白质 - 蛋白质相互作用和翻译后修饰，可确保DNA的有效快速修复，从而保护基因组完整性1 。通常，通过（共聚焦）荧光显微镜监测所谓的电离辐射诱导病灶（IRIF）的形成，研究暴露于电离辐射的细胞中的DNA修复。许多DNA修复和DNA损伤感应蛋白形成IRIF，其代表在染色质位点成核的蛋白质复合物，其维持DNA损伤2,3 。随着时间的推移，IRIF的位置和分辨率为DNA修复的时空组织提供了重要的见解，并且可能表明不同DNA修复途径的参与。损伤的DNA损伤和细胞周期阶段的性质决定了哪个DNA修复途径被激活。佛<html> r实例，在积极参与DNA复制（S期）的细胞中，同源重组（HR）是显性DNA修复途径，而在细胞周期的G1-或G2 / M期细胞中，同源末端连接（NHEJ）修复途径占优势。 DNA损伤后的最早事件之一是激活DNA损伤感应激酶共济失调血管扩张 - 突变蛋白（ATM），其主要在细胞周期的G1-和G2 / M期活跃并调节NHEJ，和共济失调血管扩张症和Rad3相关蛋白（ATR），其通过激活HR作用于S期。 ATM和ATR都是非常多效的激酶，磷酸化参与DNA修复，细胞死亡和存活的许多蛋白质4 。已经显示两种激酶在暴露于基因毒性应激后磷酸化并激活肿瘤抑制蛋白p53，表明这些激酶是关键肿瘤抑制信号轴的上游介质"xref"&gt; 5，6 。 IRIF的形成和组成通常通过使用双色免疫荧光染色和显微镜测定不同蛋白质的共定位来评估，然而，并非所有作为修复蛋白复合物的蛋白质形成IRIF，这限制了该方法的适用性。此外，（共焦）免疫荧光显微镜受光衍射性质的限制，导致约200-300nm的相当差的空间分辨率，超过大多数亚细胞结构的大小，其基本上禁止蛋白质 - 蛋白质相互作用的直接询问分子水平。因此，通过（共焦）荧光显微镜检测的免疫荧光染色模式的共定位不一定表示直接的蛋白质 - 蛋白质相互作用。最近，已经开发了新的超分辨率技术，如三维结构照相显微镜（3D-SIM） 7 ，成功应用于纳米尺度细胞研究53BP1和BRCA1 IRIF形成，揭示了共聚焦激光扫描显微镜无法检测到的这些蛋白质的空间分布特征。 可以使用其他几种方法来检测体内的蛋白质 - 蛋白质相互作用，例如共免疫沉淀，下拉法和酵母双杂交筛选方法。然而，这些技术相当繁琐，需要大量的细胞或蛋白质或涉及蛋白质的过表达，其引入实验工件。最近，已经开发了一种新技术，其允许原位 （ 即在细胞和组织中）的蛋白质 - 蛋白质相互作用的可视化和定量，称为邻近连接测定（PLA） 9,10 。镨通过与寡核苷酸（所谓的PLA探针）缀合的二级抗体检测识别感兴趣的两种蛋白质的重要​​抗体。如果两种不同的二级抗体由于被第一抗体识别的蛋白质之间的相互作用而足够接近，则缀合的寡核苷酸杂交并连接形成封闭的环状DNA底物。随后通过滚环扩增扩增此圆形底物，并用荧光染料缀合的互补寡核苷酸显色。使用PLA，蛋白质 - 蛋白质相互作用的亚细胞定位被保留，因为荧光标记的滚环扩增产物保持附着于PLA探针。基于抗体直径约为7-10nm 11的发现，该测定法的分辨率&lt;50nm。只有两对抗体（primary + secon）才能进行滚环扩增dary）在由其大小（10 + 10 + 10 + 10 = 40nm）定义的周界内物理相互作用。信号放大步骤增加了PLA测定的灵敏度，并且能够检测到几乎不表达的蛋白质的相互作用。 PLA产生可以在每个细胞基础上量化的点状，类似焦点的信号模式，由此可以评估蛋白质 - 蛋白质相互作用中的细胞内和细胞间变化。 DNA修复复合物和IRIFs的形成和组成主要在贴壁细胞系如人骨肉瘤上皮细胞系U2OS，人胚胎肾细胞系HEK293和视网膜色素上皮细胞系RPE-1中进行研究，生长和易转染。使用诸如淋巴样和骨髓细胞系的悬浮细胞培养物不太频繁，因为它们不易于转染，并且通常不粘附到盖玻片上，因此需要额外的/替代的eps成像。然而，DNA损伤的解决方案在淋巴样和骨髓恶性肿瘤的情况下非常相关，因为DNA损伤反应经常受到这些肿瘤中的基因组（驱动）畸变的影响，在正常淋巴和骨髓的恶性转化中起关键作用（祖细胞） 12,13,14 。 该协议描述了如何在悬浮细胞培养物中诱导DNA损伤后，如何使用PLA来评估和定量蛋白质 - 蛋白质相互作用。在这里，执行PLA以确定和可视化在诱导经历G1期细胞周期停滞的人B细胞白血病细胞中的DNA损伤时ATM和p53之间的相互作用。值得注意的是，本文提出的方案并不限于研究在G1停滞的白血病细胞中的ATM和p53相互作用，而且还可以用于其他蛋白质 - 蛋白质相互作用在各种细胞类型和悬浮细胞培养物中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BV173 cell line</td>
			<td>DSMZ</td>
			<td>AC-20</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>SUP-B15 cell line</td>
			<td>DSMZ</td>
			<td>ACC-389</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Gibco (Life Technologies)</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma Aldrich</td>
			<td>F7524</td>
			<td>lot #: 064M3396</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco (Life Technologies)</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco (Life Technologies)</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>imatinib methanesulfonate</td>
			<td>LC Laboratories</td>
			<td>I-5508</td>
			<td>Dissolve in DMSO, prepare 10 mM stock solution</td>
		</tr>
		<tr>
			<td>neocarzinostatin</td>
			<td>Sigma Aldrich</td>
			<td>N9162</td>
			<td>Mutagenic/teratogenic, handle with care</td>
		</tr>
		<tr>
			<td>KU55933</td>
			<td>Selleckchem</td>
			<td>S1092</td>
			<td>Dissolve in DMSO, prepare 5 mM stock solution</td>
		</tr>
		<tr>
			<td>Starfrost Microscopy Slides</td>
			<td>Waldemar Knittel</td>
			<td>VA11200 003FKB</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen liquid blocker</td>
			<td>Sigma Aldrich</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin funnel</td>
			<td>Q Path Labonord SAS</td>
			<td>003411324</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Red Starter Kit Goat/Rabbit</td>
			<td>Sigma Aldrich</td>
			<td>DUO92105</td>
			<td>Available for different species/combinations, also available in FarRED, Orange and Green</td>
		</tr>
		<tr>
			<td>goat-anti-ATM</td>
			<td>Bethyl Laboratories</td>
			<td>A300-136A</td>
			<td>PLA-grade; we succesfully used lot#A300-136A-1 in our studies</td>
		</tr>
		<tr>
			<td>rabbit-anti-phospho-Ser15-p53</td>
			<td>Cell Signaling Technology</td>
			<td>9284</td>
			<td>We succesfully used lot #9284-4 in our studies</td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Also available from various other vendors</td>
		</tr>
	</tbody>
</table>

detection and visualization of dna damage-induced protein complexes in suspension cell cultures using the proximity ligation assay

DNA损伤反应协调自发发生的DNA损伤的修复，由遗传毒性应激引起，或出现在淋巴细胞中程序性DNA断裂的上下文中。共济失调 - Telangiectasia突变激酶（ATM），ATM-和Rad3相关激酶（ATR）和DNA依赖性蛋白激酶（DNA-PKcs）的催化亚基是首先在DNA损伤诱导时被激活，并且是控制DNA修复，凋亡和细胞存活的网络的中央调节剂。作为肿瘤抑制途径的一部分，ATM和ATR通过磷酸化激活p53，从而调节p53的转录活性。 DNA损伤也导致所谓的电离辐射诱导灶（IRIF）的形成，其代表DNA损伤传感器和在DNA损伤部位积聚的修复蛋白质的复合物，其通过荧光显微镜显现。然而，蛋白质在IRIF中的共定位并不一定意味着d直接的蛋白质 - 蛋白质相互作用，因为荧光显微镜的分辨率是有限的。 原位接近连接测定（PLA）是一种新颖的技术，可以以前所未有的特异性和灵敏度直接显示细胞和组织中的蛋白质 - 蛋白质相互作用。该技术基于与感兴趣的蛋白质结合的特异性抗体的空间接近。当询问的蛋白质在〜40nm内时，扩增反应由与抗体缀合的寡核苷酸引发，扩增产物通过荧光标记显现，产生对应于相互作用蛋白质的亚细胞位置的信号。使用ATM和p53之间建立的功能相互作用作为示例，在此证明PLA如何可用于悬浮细胞培养物中以研究作为DNA损伤反应的组成部分的蛋白质之间的直接相互作用。

残留Ser15上p53的磷酸化显示依赖于ATM激酶活性16 。为了证明和证实PLA技术对悬浮细胞培养物细胞分裂素制剂的特异性，表明通过在细胞周期G1期停滞的BCR-ABL + B-ALL细胞2小时NCS处理诱导DNA损伤导致ATM和磷酸化Ser15-p53之间的特异性相互作用，如预期的那样。在用NCS处理的大多数细胞的细胞核中观察到点状PLA信号（ 图1B ），每个细胞平均?...

Watch this Scientific Journal Video about Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay at JoVE.com

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

在这里，证明了原位邻近连接测定（PLA）如何可以用于检测和可视化暴露于基因毒性应激的悬浮细胞培养物中ATM和p53之间的直接蛋白质 - 蛋白质相互作用。

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 ...

detection-visualization-dna-damage-induced-protein-complexes

Research

JoVE Journal

Biochemistry

9.9K 次观看.  Academic Medical Center, University of Amsterdam, Lymphoma and Myeloma Center Amsterdam (LYMMCARE). 在这里，证明了原位邻近连接测定（PLA）如何可以用于检测和可视化暴露于基因毒性应激的悬浮细胞培养物中ATM和p53之间的直接蛋白质 - 蛋白质相互作用。 

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

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.

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 ...

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

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

从真核细胞蛋白质复合体串联亲和纯化

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

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

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

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

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

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

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

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

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

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

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

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

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...