Name: a quantitative assay to study protein:dna interactions, discover transcriptional regulators of gene expression, and identify novel anti-tumor agents
Uploaded: 2013-08-31T07:00:00
Description: Watch this Scientific Journal Video about A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents at JoVE.c

技术援助和马里兰大学Greenebaum癌症中心转化核心设施，尤其是博士的仪器。瑞纳拉皮迪和MARIOLA Sadowska，表示诚挚的谢意。负责该实验的发展工作是由美国国立卫生研究院RO1CA108846，AHA格兰特在急救GRNT2130014，一个VA优异奖美联社报道，由马里兰州卷烟归还资金提供给马琳和斯图尔特大学（CRF）的部分资助Greenebaum癌症中心。

的时间被执行几个步骤前进，几个试剂制备和储存的过程之前：（1）细胞培养和蛋白质分离，（2）制备的寡核苷酸，（3）制备的96孔板，（4）核提取孵育过夜。程序要求，因为核蛋白质与DNA寡核苷酸孵育过夜2天。 1。缓冲液的制备 1.1核蛋白分离<ol><li>低渗缓冲液：要制备100ml的低渗缓冲液中，加入1 ml（1M的HEPES，pH值7.9），150微升（1μM的MgCl 2）?...

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	<li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nat. Protoc. 2, 1849-1861 (2007).</li><li>Bhattacharya, N., Sarno, A., Idler, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+detection+of+nuclear+factor-kappaB+activity+using+a+sensitive+oligo-based+chemiluminescent+enzyme-linked+immunosorbent+assay.">High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay.</a> Int. J. Cancer. 127, 404-411 (2010).</li><li>Hartzell, D. D., Trinklein, N. D., Mendez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+analysis+of+the+CREB+signaling+pathway+using+HaloCHIP-chip+and+high+throughput+reporter+assays.">A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays.</a> BMC Genomics. 10, 497 (2009).</li><li>Vuori, K. A., Ahlskog, J. K., Sistonen, L., Nikinmaa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TransLISA,+a+novel+quantitative,+nonradioactive+assay+for+transcription+factor+DNA-binding+analyses.">TransLISA, a novel quantitative, nonradioactive assay for transcription factor DNA-binding analyses.</a> FEBS J. 276, 7366-7374 (2009).</li><li>D'Souza, D. R., Salib, M. M., Bennett, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+Regulates+RUNX2+Activation+and+Cellular+Wound+Healing+through+the+Aldose+Reductase+Polyol+Pathway.">Hyperglycemia Regulates RUNX2 Activation and Cellular Wound Healing through the Aldose Reductase Polyol Pathway.</a> J. Biol. Chem. 284, 17947-17955 (2009).</li><li>Underwood, K. F., D'Souza, D. R., Mochin, M. T., 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=Regulation+of+RUNX2+transcription+factor-DNA+interactions+and+cell+proliferation+by+Vitamin+D3+(cholecalciferol)+prohormone+activity.">Regulation of RUNX2 transcription factor-DNA interactions and cell proliferation by Vitamin D3 (cholecalciferol) prohormone activity.</a> J. Bone Miner Res. 27, 913-925 (2012).</li><li>Held, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+ß-galactosidase+activity+using+the+PowerWave+HT+and+Gen5+data+analysis+software:+basic+enzyme+kinetic+determinations.">Kinetic analysis of ß-galactosidase activity using the PowerWave HT and Gen5 data analysis software: basic enzyme kinetic determinations.</a> Biotek Instruments Application Note. , (2007).</li><li>Renard, P., Ernest, I., Houbion, 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=Development+of+a+sensitive+multi-well+colorimetric+assay+for+active+NFkappaB.">Development of a sensitive multi-well colorimetric assay for active NFkappaB.</a> Nucleic Acids Res. 29, E21 (2001).</li><li>Pommier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+topoisomerase+I+inhibitors:+chemistry,+biology,+and+interfacial+inhibition.">DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition.</a> Chem Rev. 109, 2894-2902 (2009).</li><li>Pennisi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics.+ENCODE+project+writes+eulogy+for+junk+DNA.">Genomics. ENCODE project writes eulogy for junk DNA.</a> Science. 337, 1159-1161 (2012).</li></ol>

DNA结合测定法来测量的转录因子与DNA的相互作用的能力。检测DNA结合包括电泳迁移率（EMSA）1和染色质immuneprecipitation（芯片）为基础的分析3以及检测采用96孔格式4，如化学发光检测2。该EMSA是非定量和使用放射性标记（32 P）的寡核苷酸。这些孵育核蛋白质和结合复合体上分离的琼脂糖或聚丙烯酰胺凝胶。与此相反，定量的D-ELISA是能够测量对应于在RUNX2靶?...

DNA结合分析在测量转录因子与DNA相互作用的能力效用。检测DNA结合包括电泳迁移率变动分析（EMSA）依赖于放射性标记的寡核苷酸1或化学发光检测2。染色质immuneprecipitation（芯片）为基础的分析3以及测定使用96孔格式4也被描述。然而，EMSA是一个非定量测定要求使用放射性标记的寡核苷酸。当核的蛋白质相关联的特定核苷酸序列的启动子，结合复合体是滞后于聚丙烯酰胺凝胶和特定的转录因子可与抗体“超迁移”进行验证。我们已经开发出利用酶联免疫格式（D-ELISA），它能够测量与对应于所定义的启动子元件的DNA结合序列，RUNX2的相互作用的定量DNA结合测定法我Ñ​​RUNX2靶基因。使用防RUNX2抗体提供了特异性的检测和缺乏放射性标记的从传统的凝胶迁移实验5分辨这个实验。检测结合复合体是可能的使用结合到辣根过氧化物酶（HRP），它的HRP底物四甲基联苯胺（TMB）转化成有色产物进行分光光度分析的二次抗体。本文报道的测定可以合并使用突变的DNA的寡核苷酸作为对照，并可以用于检测的DNA的竞争性或非竞争性抑制剂结合。新颖的抗肿瘤化合物的筛选，也可以用此试验。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Poly dI/dC</td>
			<td>GE Healthcare, Piscataway, NJ</td>
			<td>US20539-5UN</td>
			<td>1 U ~50mg</td>
		</tr>
		<tr>
			<td>RUNX2 antibody</td>
			<td>MBL International Corp., Woburn, MA</td>
			<td>D130-3</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Fab-specific peroxidase conjugated antibody</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A9917</td>
			<td>7.1 mg/ml</td>
		</tr>
		<tr>
			<td>TMB Substrate (tetramethyl benzidine)</td>
			<td>EXALPHA Biologicals, Shirley, MA</td>
			<td>X1189S</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>57995</td>
			<td>Plate-fixing</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>VWR, West Chester, PA</td>
			<td>BDH-39922-1</td>
			<td>Stop solution</td>
		</tr>
		<tr>
			<td>Multi-well plates</td>
			<td>Greiner Bio-One, Basel, Switzerland</td>
			<td>655996</td>
			<td>Avidin-coated, black sides</td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>Thermo-Scientific/Pierce, Rockford, IL</td>
			<td>78440</td>
			<td>Protease and phosphatase inhibitors</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td>Various manufacturers</td>
			<td>Laboratory grade</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 1. Reagents</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Biotrak II Visible plate reader</td>
			<td>Amersham Biosciences</td>
			<td>&nbsp;</td>
			<td>For use with stop reaction method</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Bio-Tek Synergy HT Multi-reaction microplate reader</td>
			<td>Bio-Tek Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>For use with continuous kinetic monitoring</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Equipment</td>
		</tr>
	</tbody>
</table>

a quantitative assay to study protein:dna interactions, discover transcriptional regulators of gene expression, and identify novel anti-tumor agents

许多DNA结合测定法，如电泳迁移率变动分析（EMSA），化学发光测定法，染色质免疫沉淀（ChIP）为基础的检测，和多孔基测定法用于测量转录因子的活性。然而，这些测定是非定量，缺乏特异性，可能涉及使用放射性标记的寡核苷酸，并且可能并不适用于DNA结合的抑制剂的筛选。另一方面，用一个定量的DNA结合的酶联免疫吸附测定法（D-ELISA）测定中，我们展示了核蛋白质相互作用与DNA的使用依赖于特定的关联与存在于生物素共有DNA结合序列的RUNX2转录因子标记的寡核苷酸。细胞的制备，提取核蛋白，和双链寡核苷酸的设计进行说明。抗生物素蛋白包被的96孔板中是固定的碱性缓冲液，温育在核苷酸封闭缓冲液中的核蛋白质。傅杰仕由于板的彻底清洗，特定的一抗和二抗孵育，随后加入比色反应的辣根过氧化物酶底物和发展。停止反应模式或连续动力学监测被用来定量测量与DNA蛋白相互作用。我们讨论了相应的特异性控制，包括与非特异性IgG或无蛋白质或初级抗体治疗。该测定的申请中描述，包括其在药物筛选程序和代表正和负的结果进行了讨论。

对D-ELISA法是高度特异性的针对指定的DNA结合蛋白，只要使用含有共识RUNX2结合位点（ACACCA）的三个拷贝的序列特异性双链寡核苷酸。第一抗体识别的蛋白因子也提高了特异性。二次抗体包含共价连接的辣根过氧化物酶（HRP），一个透明基底（四甲基联苯胺）转换为有色产物，便于检测（ 图1）。出于这些原因，一些重要的背景控制需要被包括在每个测定板，以确保特定的DNA结合。这些...

Watch this Scientific Journal Video about A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents at JoVE.c

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

我们开发了一种定量的DNA结合，基于ELISA的测定法来测量与DNA的转录因子相互作用。高特异性的RUNX2蛋白与共识的DNA寡核苷酸识别和特异性单克隆抗体来实现。比色检测用酶偶联抗体底物反应的实时监测。

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...