Name: bimolecular fluorescence complementation (bifc) assay for protein-protein interaction in onion cells using the helios gene gun
Uploaded: 2010-06-12T17:00:00
Description: Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun at JoVE.com

<p class="jove_content">我们感谢博士。史蒂夫和晓宁张山的35S：：GFP pGlowbug构造的Hokensen Z。欧莱雅的实验室研究到CH的博士后研究是由美国国家科学基金会（IOB0616096和MCB0744752）支持。 ZL是由美国马里兰大学农业试验站的部分支持。 </p>

<p class="jove_title">一，质粒制备</p><p class="jove_content">轰击使用的质粒DNA必须是高纯度和浓度500ng/μl或更大。附设涉及两个质粒结构，需要25微克的每个质粒。换句话说，两个质粒的1:1摩尔比混合，给墨盒准备上升​​到50微克质粒DNA总量。要留意超过50微克，增加更多的DNA可能会导致金颗粒的集聚，并应避免。市售的质粒DNA提取试剂盒隔离质粒DNA的建议。 </p><p class="jove_content">质粒载体应在pUC19中，如规模相对较小。?...

<ol>
	<li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> <em style='font-style: italic'>Mol Cell</em>. <b>9</b> (4), 789-789 (2002).</li><li>Sitaraman, J., Bui, M., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LEUNIG_HOMOLOG+and+LEUNIG+perform+partially+redundant+functions+during+Arabidopsis+embryo+and+floral+development.">LEUNIG_HOMOLOG and LEUNIG perform partially redundant functions during Arabidopsis embryo and floral development.</a> <em style='font-style: italic'>Plant Physiol</em>. <b>147</b> (2), 672-672 (2008).</li><li>Walter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> <em style='font-style: italic'>Plant J</em>. <b>40</b> (3), 428-428 (2004).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> <em style='font-style: italic'>J Vis Exp</em>. , (2008).</li><li>Azhakanandam, S., Nole-Wilson, S., Bao, F., Franks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEUSS+and+AINTEGUMENTA+mediate+patterning+and+ovule+initiation+during+gynoecium+medial+domain+development.">SEUSS and AINTEGUMENTA mediate patterning and ovule initiation during gynoecium medial domain development.</a> <em style='font-style: italic'>Plant Physiol</em>. <b>146</b> (3), 1165-1165 (2008).</li><li>Zhang, X. N., Mount, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+alternatively+spliced+isoforms+of+the+Arabidopsis+SR45+protein+have+distinct+roles+during+normal+plant+development.">Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.</a> <em style='font-style: italic'>Plant Physiol</em>. <b>150</b> (3), 1450-1450 (2009).</li></ol>

<ol><li>对于首次用户，我们建议实行整个过程，从墨盒准备观察，用一个单一的质粒构记者如35S：：GFP或35S：：GUS。我们使用了35S：：GFP质粒从博士获得。史蒂夫山和小宁张<sup>6（</sup>图1A，D）。不适合植物基因出手控制RioRad（猫165-2244）出售的墨盒。 </li><li>对于附设，第二个阳性对照是必要的，应当由两个已知的交互融合YFP的C端和N -末端片段的合作伙伴，分别。 </li><li>对于附设，这是必要的，包括一些负面控制...

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		<th>Catalogue Number</th>
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<td>Yellow onion</td>
<td>&nbsp;</td>
<td>Any supermarket</td>
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<td>Helios Gene Gun</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>&nbsp;</td>
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<td>Fluorescent microscope with appropriate filters for GFP or YFP</td>
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<td>Zeiss, Nikon, Olympus</td>
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bimolecular fluorescence complementation (bifc) assay for protein-protein interaction in onion cells using the helios gene gun

<p class="jove_content">在不同生物体的基因功能的研究依赖于基因产物如何彼此互动在正常的细胞环境的知识。双分子荧光互补（附设）含量<sup&gt; 1</sup&gt;，使研究人员能够可视化在活细胞内蛋白质相互作用，并已成为一个重要的研究工具。此法是基于两个片段的一种荧光蛋白（GFP）的，每一个融合到一个潜在的相互作用蛋白伙伴促进协会。两个蛋白合作伙伴的互动，将有利于协会的N端和C -末端片段的GFP，导致荧光。对于植物的研究人员，洋葱表皮细胞是一种理想的实验系统进行，因为在获得和准备洋葱组织的易用性和最小的背景荧光的荧光直接​​可视化的附设实验。赫利奥斯基因枪（BioRad公司）是常用的轰击洋葱细胞的质粒DNA。我们证明使用赫利俄斯基因枪引入两个相互作用的质粒结构<em&gt;拟南芥</em&gt;转录因子，苏斯（SEU）和LEUNIG同源（LUH）<sup&gt; 2</sup&gt;和它们之间的相互作用附设在洋葱表皮细胞介导的​​可视化。</p>

Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun at JoVE.com

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

<p class="jove_content">本文介绍了如何正确使用BioRad公司赫利奥斯基因枪引入洋葱表皮细胞的质粒DNA，以及如何测试基于双分子荧光互补的原则（附设）洋葱细胞中的蛋白质 - 蛋白质相互作用</p>

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Research

JoVE Journal

Biology

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Gene-gun Transfection of Hippocampal Neurons

海马神经元的基因枪转

科研

生物学

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

基因枪子弹的制备和神经元的基因枪转染切片文化

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Bimolecular Fluorescence Complementation

双分子荧光互补

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...