我们感谢SS Wang，Amar和A. Galla对手稿的批判性阅读。本出版物报道的研究得到了美国国家卫生研究院综合医学科学研究所的支持，获得了授权号RO1GM067837和RO1GM056006（SAK）。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方观点。

<html> 1.构建和记录质粒制备<ol><li> PCR扩增待分析的靶DNA的启动子区/"片段"（优选约250-500bp），以使用大肠杆菌 （TOP10）进一步克隆入入载体。 </li><li>一旦测序确认，亚克隆阳性克隆在7如前所述8，9，10，11兼容pGLacZi表达载体。 </li><li>通过pGLacZi的同源重组变换启动子融合（YM4271）的酵母菌株来产生酵母报告菌株如前面12,13说明。这里没有描述具有DNA区域的酵母菌株与培养基配方的转化和确认步骤，因为步骤类似于经典的Y1H方案屁股= "外部参照"&gt; 9，10，11，12，13。所述PEXP-AD502对照质粒可以同时quantifiying如前所述8β半乳糖苷酶活被用于归一化目的。 </li><li>克隆感兴趣的TF或蛋白质，随后将相互作用的蛋白质伴侣克隆到pDEST22和pDEST32ΔDBD（pDEST32减去DNA结合域）表达载体中。转化的酵母启动子片段和TF克隆随后在方案中进一步使用。 </li></ol> 2.修改Y1.5H协议<ol><li>在第1天（下午），从培养皿或甘油浆中的SD-Ura中的 5mL培养物开始，并在30℃振荡器中孵育过夜。 注意:这种培养可以从新鲜条纹板或直接从酵母的25％或30％甘油原料开始用DNA片段克隆。 </li><li>在第2天（下午），用500μL过夜培养物接种10mL YPDA培养基，并在30℃振荡器中孵育过夜。 </li><li> 第3天（清晨和整个下午）:准备感受态细胞（清晨）。 <ol><li>用2 mL过夜培养物接种100mL的YPDA培养基，并在30°C振荡器中孵育。生长这种新鲜的培养物，直到OD 600达到0.4-0.8（3-5小时）。 注意:检查过夜培养物的OD 600 ，并确保此步骤的起始点OD 600为0.1 - 0.2。使用过度培养或培养不足的培养物可能会延长实验。 </li><li>在1000xg和21℃下离心5分钟。丢弃上清液。 </li><li>使用旋涡或上下移液将颗粒重建在10mL无菌水中。 </li><li>在1000xg和21℃下离心5分钟。丢弃上清液。 </li><li> Reconstitu使用涡旋或吸管上下游，在2mL Tris-乙二胺四乙酸（TE）/乙酸锂（LiAc）中沉淀。 注意:请参见表1中 TE / LiAc的配方。 </li><li>离心5分钟1000 xg和21°C。丢弃上清液。 </li><li>在4 mL TE / LiAc + 400μL鲑鱼精DNA（10 mg / mL）中上下重复悬浮沉淀，并进行下一步。 </li></ol></li><li> 共同转化:热休克细胞（下午晚些时候） <ol><li>在96孔混合板（U底）中分配每孔20μL的细胞。 </li><li>向孔中加入每个pDEST22:TF质粒，pDEST32ΔDBD:TF质粒和pEXP-AD502加pDEST32ΔDBD:TF（归一化对照）150ng（〜1.5μL） 注意:为了成功共同转化，pDEST22:TF和pDEST32ΔDBD:TF质粒各约150ng，预期最佳结果。可以随构建体和质粒表达而变化需要每个实验进行优化。另外一个控件pDEST22-TF加pDEST32deltaDBD也可以由用户自行决定。 </li><li>每孔加入100μLTE / LiAc /聚乙二醇（PEG）（ 混合三次 ）。 注意:请参见表1中 TE / LiAc / PEG的配方。 </li><li>用透气密封盖住板，并在30℃下孵育20-30分钟（可以更长时间）（无需搅拌）。 </li><li>在42℃下热冲击20分钟（ 精确 ）。 </li></ol></li><li> 平板细胞 <ol><li>在1000xg和21℃下离心5分钟。使用多通道移液器去除上清液。加入110μLTE，混匀。 </li><li>在1000xg和21℃下离心5分钟。使用多通道移液管去除100μL上清液。用打孔机重新悬挂颗粒。 </li><li>将细胞转移到SD-Trp-Leu琼脂平板上。孵化板在30°C直到下一步。 </li></ol></li><li> 第5天（下午）:使用打孔机/微孔板复制器将细胞转移到新板上。 <ol><li>使用多通道移液管，用无菌的96孔混合板（U底）填充50μLTE。在TE中预冲洗打孔机。 注意:打孔机或微孔板复制器应在此步骤之前进行灭菌，随后在方案中进行灭菌。可以应用将浸入乙醇（100％，不分子生物学级），然后将其燃烧到火焰上的冲孔机的常见方法。等待1分钟冷却冲孔针。 注意:如果在长凳上进行灭菌;确保工作台用乙醇预先清洁，周围没有任何可燃物质。穿戴适当的个人防护装备（PPE）。 </li><li>打开殖民地并在TE填充的板块中重新挂起。 注意:如果平板生长平稳，可以直接打孔到TE填充板或替代地，应使用消毒牙签将单个菌落（在多个菌落的情况下）捡拾到TE填充的板上，随后打孔机可以用于下一步骤。 </li><li>转移到新的SD-Trp-Leu琼脂平板上，以获得最佳的表面覆盖率。孵育板在30°C直到下一步。 </li></ol></li><li> 第7天（下午）:开始培养以测定β-半乳糖苷酶活性。 <ol><li>使用多通道移液管，用无菌96孔混合板（U底）填充50μL的SD-Trp-Leu培养基。 </li><li>使用多通道移液管，用180μL的SD-Trp-Leu培养基填充无菌96-深孔块。 </li><li>将培养基中的冲孔机预先浸湿，并将菌落从板转移到50μL的培养基。 </li><li>将20μL酵母转移到180μL的SD-Trp-Leu培养基中，并用透气密封件密封块。孵化在30℃振荡器下36小时。 </li></ol></li><li> 第9天（早晨）:开始用于酶测的培养。 <ol><li>将100μL的培养物转移到无菌96深孔块中的500μLYPDA培养基中。 </li><li>用透气密封盖住灭菌的深孔板，并在30℃下以200rpm搅拌孵育3-5小时（直至OD 600 0.3-0.6）。 </li><li>使用多通道移液管在分光光度计平板上重新悬浮培养并转移125μL（测量OD 600 ）。 注意:注意:如果OD 600低于0.3 - 0.6，请勿分配最后一滴以避免气泡，请根据读数将剩余的细胞孵育1 - 2小时。在此步骤中使用的125μL培养基可以丢弃或转移回原始的深井区块，用户可以自行决定。 </li><li>在3000×g和21℃下离心深井块中剩余的细胞10分钟。去除上清液t反转。 </li><li>加入200μLZ缓冲液并涡旋。 注意:请参见表1中 Z缓冲区的配方。 </li><li>在3000 xg和21℃下离心5分钟。倒置去除上清液。 </li><li>加入20μLZ缓冲液和涡旋。 </li><li>用可以抵抗冻融循环的密封箔覆盖板。在通风柜中使用液氮进行4个循环的冷冻/融化，然后在水浴中进行42℃（每次2分钟）。 </li><li>加入200μLZ-缓冲液/β-巯基乙醇（β-ME）/邻硝基苯基-β-半乳糖苷（ONPG）（12mL，21μL，8.4mg分别得到20mL溶液; 总是制备新鲜的 ）。 注意:ONPG需要一些时间才能正确溶解，以便在达到此步骤前一小时准备溶液。 ONPG作为测定β-半乳糖苷酶活性的底物。 </li><li>在30°C下孵育高达17 - 24小时（如果颜色发展较早，请检查继续下一步）。 </li></ol></li><li> 第10天（早晨）:停止反应并测量。 <ol><li>加入110μL1M Na 2 CO 3以停止反应并记录时间。 </li><li>涡旋并在3000 xg和21℃下离心10分钟。 </li><li>使用多通道取125μL上清液并测量OD 420 。 注意:注意，不要分配最后一滴以避免气泡。 </li><li>在电子表格中计算β-gal活性:1000×OD 420 /（时间（分钟）×体积（mL）×OD 600 。 </li></ol></li></ol>

<ol>
	<li>Saghbini, M., Hoekstra, D., Gautsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Media+formulations+for+various+two-hybrid+systems.">Media formulations for various two-hybrid systems.</a> Methods Mol Biol. 177, 15-39 (2001).</li><li>Reece-Hoyes, J. S., Marian Walhout, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+one-hybrid+assays:+A+historical+and+technical+perspective.">Yeast one-hybrid assays: A historical and technical perspective.</a> Methods. 57 (4), 441-447 (2012).</li><li>Rigaut, G., 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=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Puig, 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=The+Tandem+Affinity+Purification+(TAP)+Method:+A+General+Procedure+of+Protein+Complex+Purification.">The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Gavin, A. -. C., 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=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415 (6868), 141-147 (2002).</li><li>Tripathi, P., Carvallo, M., Hamilton, E. E., Preuss, S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+B-BOX32+interacts+with+CONSTANS-LIKE3+to+regulate+flowering.">Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering.</a> Proc Natl Acad Sci USA. 114 (1), 172-177 (2017).</li><li>Helfer, A., 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=LUX+ARRHYTHMO+encodes+a+nighttime+repressor+of+circadian+gene+expression+in+the+Arabidopsis+core+clock.">LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock.</a> Curr Biol : CB. 21 (2), 126-133 (2011).</li><li>Pruneda-Paz, J. L., 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=A+genome-scale+resource+for+the+functional+characterization+of+Arabidopsis+transcription+factors.">A genome-scale resource for the functional characterization of Arabidopsis transcription factors.</a> Cell Rep. 8 (2), 622-632 (2014).</li><li>Wanke, D., Harter, K. <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+Plant+Regulatory+DNA+sequences+by+the+Yeast-One-Hybrid+Assay.">Analysis of Plant Regulatory DNA sequences by the Yeast-One-Hybrid Assay.</a> Methods Mol Biol. 479, 291-309 (2009).</li><li>Klein, P., Dietz, K. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-Binding+Proteins+and+Protein-Protein+Interactions+by+Yeast+One-Hybrid+and+Yeast+Two-Hybrid+Screen.">Identification of DNA-Binding Proteins and Protein-Protein Interactions by Yeast One-Hybrid and Yeast Two-Hybrid Screen.</a> Methods Mol Biol. 639, 171-192 (2010).</li><li>Breton, G., Kay, S. A., Pruneda-Paz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Arabidopsis+Transcriptional+Regulators+by+Yeast+One-Hybrid+Screens+Using+a+Transcription+Factor+ORFeome.">Identification of Arabidopsis Transcriptional Regulators by Yeast One-Hybrid Screens Using a Transcription Factor ORFeome.</a> Methods Mol Biol. 1398, 107-118 (2016).</li><li>Deplancke, B., Vermeirssen, V., Arda, H. E., Martinez, N. J., Walhout, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway-Compatible+Yeast+One-Hybrid+Screens.">Gateway-Compatible Yeast One-Hybrid Screens.</a> CSH Protoc. 2006 (5), (2006).</li><li>Deplancke, B., Dupuy, D., Vidal, M., Walhout, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gateway-compatible+yeast+one-hybrid+system.">A gateway-compatible yeast one-hybrid system.</a> Genome Res. 14 (10B), 2093-2101 (2004).</li></ol>

作者声明没有利益冲突。

现有的Y1H标准方法适用于鉴定与其DNA诱饵结合的单一蛋白质猎物。通过各种技术修改，现有系统已被用于定义转录调控网络。然而，TF已知作为涉及两种或更多种TF或蛋白质的复合物的一部分，只有一些TF能够结合DNA。仅具有蛋白质结合结构域并且缺乏与DNA结合能力的蛋白质或TF更可能在复合物中起作用，优选使用能够结合DNA的TF。使用传统Y1H的高通量屏幕偶尔会忽略这些生物重要的相互作用。因此...

<html>一般地，为了了解蛋白质-DNA相互作用，所述Y1H测定是在实验室设置1成功地使用的优选系统。基本的Y1H测定涉及两个组分:a）在编码报道蛋白的基因上游成功克隆了目标DNA的报道构建体;和b）将在感兴趣的TF和酵母转录激活结构域（AD）之间产生融合蛋白的表达构建体。感兴趣的DNA通常被称为"诱饵"，而融合蛋白被称为"猎物"。在过去几年中，已经开发了多种版本的Y1H测定法，以适应具体需求，具有自己的优点2 。可以成功实施现有的Y1H测定，以一次鉴定和验证一种蛋白质与其DNA诱饵的相互作用，但缺乏识别异二聚体或多聚体蛋白质 - DNA相互作用的能力。 "&gt;这里描述的方法是现有Y1H的修改版本，使研究人员能够同时研究与其靶DNA序列结合的多种蛋白质，该测定的目的是用激活结构域表达感兴趣的蛋白质（pDEST22: TF），并在酵母系统中存在和/或不存在相互作用的蛋白质配偶体（pDEST32ΔDBD-TF）的情况下评估与报告子融合的候选DNA区域的活化，该测定将允许我们确定这两个蛋白质是激活靶标所必需的，这是一种GATEWAY克隆兼容系统，因此可用于常规实验室设置，该方案基于直接转化，提供了酵母系统中不同TFs的共转化因此，验证蛋白质复合物与其可能的DNA靶标结合的体外分子和功能验证是一个有利的策略或任何系统。如串联亲和纯化（TAP）的方法目前的技术是昂贵的和劳动密集的，但允许大规模3，4，5蛋白hetrocomplexes的检测。这种修饰的酵母单杂交体可以在小规模的常规实验室设置中成功进行（ 图1 ），并且也具有成本效益。 Y1.5H测定可以促进社区的研究人员测试和验证他们的假设，以使用异源系统定义多聚体蛋白复合物与常见DNA靶标的作用。根据研究结果，该假设可以在优选的研究系统中在体内得到验证。最近，我们采用这种方法来定义TF的作用及其作用方式来调节拟南芥6的开花。

<table border="0" cellpadding="0" cellspacing="0" width="846">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pDEST22 vector</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">PQ1000101</td>
			<td style="width:332px;">Pro-Quest Two hybrid system kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pDEST32delatDBD</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">PQ1000102</td>
			<td>Pro-Quest Two hybrid system kit; this vecor is modified. It is pDEST32 minus DNA binding Domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pENTR/D-TOPO Cloning Kit</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">K240020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">YM4271 Strain</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">K1603-1</td>
			<td>MATCHMAKER One-Hybrid System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">pEXP-AD502</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">PQ1000101</td>
			<td>Pro-Quest Two hybrid system kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">GATEWAY LR Clonase II enzyme mix</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">11791020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">YPDA media</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">SD-Agar</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">SD minimal media</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Uracil DO Supplement</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Tryptophan Do Supplement</td>
			<td style="width:132px;">Clontech</td>
			<td style="width:157px;">630413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Tris Base</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">EDTA</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">S311-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">LiAc</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">L4158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Saplmon Sperm (10mg/ml)</td>
			<td style="width:132px;">Invitrogen</td>
			<td style="width:157px;">15632011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">96 well round bottom Plate</td>
			<td>Greiner bio-one</td>
			<td align="right">650101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">PEG3350</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">1546547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">96-deep well block</td>
			<td style="width:132px;">USA Scientific</td>
			<td style="width:157px;">1896-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Sealable Foil</td>
			<td style="width:132px;">USA Scientific</td>
			<td style="width:157px;">2923-0110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Araseal</td>
			<td style="width:132px;">Excel Scientific</td>
			<td style="width:157px;">B-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">2-mercaptoethanol</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">034461-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">ONPG</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">73660</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">223484</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">S3264</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">S3139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">KCL</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">BP366-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4&#160;</td>
			<td style="width:132px;">Sigma-Aldrich</td>
			<td style="width:157px;">83266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">HCL</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:157px;">SA54-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Drybath</td>
			<td style="width:132px;">Thermo Fischer</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Voretx</td>
			<td style="width:132px;">Thermo Fischer</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Centrifgue</td>
			<td style="width:132px;">Eppendorf</td>
			<td style="width:157px;">Centrifuge 5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Plate Reader</td>
			<td style="width:132px;">Molecular Device</td>
			<td style="width:157px;"></td>
			<td>SPECTRAMAX PLUS Microplate Spectrophotometer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:225px;">Incubator</td>
			<td style="width:132px;">Thermo Fisher</td>
			<td style="width:157px;">Model No. 5250- 37 Degree, 6250-30 degrees</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Shaker Incubator</td>
			<td style="width:132px;">New Brunswick</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Water Bath</td>
			<td style="width:132px;">Thermo Fisher</td>
			<td style="width:157px;">IsoTemp 205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Puncher</td>
			<td style="width:132px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">50 ml Falocn</td>
			<td style="width:132px;">BD Falcon</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Beaker</td>
			<td style="width:132px;">Nalgene</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Flask</td>
			<td style="width:132px;">Nalgene</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Petriplates (150mm)</td>
			<td style="width:132px;">Greiner bio-one</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified yeast-one hybrid system for heteromeric protein complex-dna interaction studies

多年来，酵母单杂交测定已被证明是识别和验证蛋白质（如转录因子（TF））及其DNA靶标之间的物理相互作用的重要技术。本文提出的方法利用Y1H的基本概念，但进一步修改以研究和验证与其靶DNA结合的蛋白质复合物。因此，其被称为修饰的酵母单杂交（Y1.5H）测定。该测定是成本有效的，并且可以在常规实验室设置中容易地进行。 尽管使用异源系统，但是所描述的方法可能是用于在任何研究系统，特别是植物基因组学中测试和验证异源蛋白复合物与其用于功能基因组学的DNA靶标结合的有价值的工具。

用于共同转化酵母细胞中DNA区域与感兴趣蛋白质的一般平板安装程序（ 图2 ）。可以根据实验的需要和测试的DNA区域/片段的数量来修改平板设置。在方案的第5天之后， 如图3所示，可以看到良好生长和阳性的平板。在我们的研究中，从转录起始位点开始上游的2600bp的启动子区被分成6个区域或片段（FR 1-6） 6 ?...

Watch this Scientific Journal Video about A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies at JoVE.com

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

这里描述的修饰的酵母单杂交测定法是经典酵母单杂交（Y1H）测定法的扩展，以研究和验证异源蛋白复合物 - DNA异构体系中任何功能基因组学研究的相互作用。

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

a-modified-yeast-one-hybrid-system-for-heteromeric-protein-complex

Research

JoVE Journal

Biochemistry

11.3K 次观看.  University of Southern California. 这里描述的修饰的酵母单杂交测定法是经典酵母单杂交（Y1H）测定法的扩展，以研究和验证异源蛋白复合物 - DNA异构体系中任何功能基因组学研究的相互作用。 

视频: A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

重组蛋白表达，结晶和生物物理研究<em&gt;芽孢杆菌</em&gt; - 保守的核苷酸焦磷酸化酶，BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to 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 ...

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 ...

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 ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

使用χCRAC以高时间分辨率监测体内蛋白质-RNA相互作用动力学

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

用于异构蛋白复合物 - DNA相互作用研究的修饰的酵母一杂交系统

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