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。

一，质粒制备轰击使用的质粒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。 质粒载体应在pUC19中，如规模相对较小。我们克隆到的pUC19 - SPYNE的pUC19 - SPYCE，分别为3的cDNA SEU和LUH。 农杆菌介导的二进制向量过大和不适当的轰击。 二。墨盒准备墨盒准备涉及涂料与质粒DNA和装入塑料管材，随后削减到半英寸长片，它可以加载到基因枪匣持有的金粒子。墨盒准备在一个单独的朱庇特第4条的详细描述，这里没有描述。洋葱细胞的轰击，我们建议12.5毫克每准备金颗粒，产量约在1微克DNA/0.25毫克黄金/盒，50墨盒50微克质粒DNA混合。每个墨盒是一炮打响。金颗粒可以是0.6微米，直径1.0微米（猫＃1652262或Cat＃1652263; BIORAD）。我们还建议使用的墨盒准备为0.5毫克/毫升PVP的解决方案。对于附设，结合每个公认的互动合作伙伴，在相同的墨盒准备的两个质粒DNA。 墨盒准备后，重要的是要测试发射在150到200 psi之间的氦气压力培养皿周围包裹成方形的封口膜，新提出的墨盒墨盒。这将测试如果金粒子可以有效地推动墨盒，你应该看到微弱的金颗粒上的封口膜。这使您每次拍摄将覆盖的区域的想法。您可能会注意到不同数量的金颗粒上的封口膜在不同氦气压力（150-200 psi范围）的推动。如果没有显着差异，选择压力较低的轰击。 三。准备洋葱组织在拍摄当天，从一个大的黄皮洋葱的外层皮肤剥离。使用清洁和锋利的刀片，切4-5层约2 x 8厘米深的矩形区域。取出的矩形洋葱组织，放弃外两层，其余各层的洋葱切成约2X1.5厘米件。放置在培养皿滤纸盘（削减成圆圈的Whatman 3MM纸）洋葱片，用无菌水浸湿。盖培养皿中，他们已经准备好去。 四。轰击<ol><li>墨盒装入白色圆形盒持有人。应持有不同的墨盒装入含不同质粒结构的墨盒。每个墨盒持有人最多可容纳12墨盒为12杆。 </li><li>插入电池和新鲜桶内胆的Helios基因枪。确保桶内衬和枪有各自的O型圈连接。 </li><li>首先，插入一个空盒支架，基因枪与马克＃12墨盒上面临持有枪顶部。预先盒支架，一次或两次，以确保它是正确插入。 </li><li>附加枪氦坦克和调整的压力在150至200磅之间。 </li><li>穿耳朵的保护和消防枪持有空墨盒几次清理出枪室，并确保压力稳定。 </li><li>取出空墨盒的持有人，并插入持有装入墨盒。提前盒支架，使你想火墨盒墨盒持有人（6点）定位在底部。洋葱片在这里拍摄，提供了一个背景荧光阴性对照的形式。 </li><li>洋葱片放置在一个空的最内层朝上洋葱层的培养皿中。休息基因枪的培养皿桶内衬和消防枪扣动扳机的同时，按住侧面按钮。传输轰击洋葱含有湿润3MM滤纸的培养皿中。 </li><li>前进到下一个墨盒和火灾不同的洋葱片。重复此步骤，直到四，五个洋葱片拍摄。每个洋葱片拍摄一次。 </li><li>变更与含不同质粒或不同质粒组合的墨盒装入不同的墨盒持有人。请记住一个改变桶内衬小号以及防止污染。拍四，五个洋葱片。 </li><li>轰击后，返回所有的洋葱perti料理。盖有盖培养皿。包装用封口膜，以防止干燥。在16-48小时的黑暗，在室温下孵育洋葱片。 </li><li>关闭之前从氦气罐分离基因枪的气体，并释放压力。拧开桶内衬，并取出墨盒持有和使用（或闲置）墨盒。 </li><li>浸没在烧杯用肥皂水清洗墨盒持有人及桶内衬。在洗澡sonicator的烧杯中，超声20分钟。之后，用清水冲洗干净，除去所有肥皂残留物。 70％的乙醇浸泡一个小时，然后干纸巾。 </li></ol>五，观察<ol><li>孵化轰击洋葱件在室温为16-20小时后，我们可以使用蔡司Axio上Observer.z1如在这项研究中所使用的荧光显微镜观察GFP或YFP的荧光。 </li><li>使用镊子慢慢剥过的洋葱内表皮（层，直接面对在轰击枪）的单细胞层平面两端。放置到幻灯片上的水下降剥离。加盖玻片，但要小心，尽量减少气泡。 </li><li>明场显微镜下，首先检查细胞质流，以确保细胞是活的的。要做到这一点，揭露幻灯片明视场光几分钟热身洋葱组织，然后寻找移动质。细胞质流并不总是很容易看到，所以不要气馁，如果你不能看到任何。 </li><li>荧光检测，使用适当的激发和检测GFP或YFP过滤器。这一点很重要，包括阴性对照，如洋葱片拍摄与非荧光质粒。微弱的黄色信号，可能会出现受伤的细胞，气泡，或碎片。同样重要的是，包括阳性对照。一个组成型表达质粒含有荧光报告，如35S：：GFP是一个很好的的阳性对照。可见GFP的洋葱表皮细胞（图1A，D）的信号表明，墨盒，洋葱组织和基因枪射击适当的准备和执行。随后，我们观察到SEU - pSPYNE LUH - pSPYCE之间的相互作用，包含了35S的混合物墨盒轰击洋葱片：SEU - pSPYNE和35S：：LUH - pSPYCE的DNA。淡淡的YFP的荧光观察（图1 B，E），这表明仅在细胞核的SEU和LUH物理之间的直接互动，带来了在细胞核分裂YFP的片段。请注意，从附设的YFP的信号明显高于GFP信号较弱，从35S：：GFP。 </li></ol>代表性的成果在这里我们的研究报告，35S：：GFP阳性对照质粒，充满整个细胞，包括细胞核和细胞质（图1A，D）的散发出强烈的荧光。 GFP蛋白是足够小，没有一个核定位信号核弥漫。相比之下，SEU和LUH两个拟南芥转录因子先前交互显示在酵母双杂交实验2。 SEU - pSPYNE（SEU的YFP的N -端片段融合）LUH - pSPYCE（SEU的YFP的N -端片段融合），扩散到细胞核中，含有核定位信号2,5太大。 YFP的荧光信号，在洋葱细胞核（图1 B，E）检测表明，SEU - pSPYNE LUH - pSPYCE可以在洋葱细胞核交互。无荧光信号检测与载体含有pSPYNE YFP的LUH - pSPYCE（图1C，F）的N -端片段的质粒混合轰击洋葱。 附设介导的YFP的荧光明显高于从35S绿色荧光蛋白介导的的荧光较弱：GFP质粒。在我们的研究中，他们也表现出不同的亚细胞定位（与B比较图1A）。此外，荧光细胞数为附设明显降低，为附设只有当两个伙伴的质粒成功进入，并表示在相同的洋葱细胞。 <img src="/files/ftp_upload/1963/1963fig1.jpg" alt="图1" /> 图1。荧光灯（A，B，C），和明场（D，E，F）的洋葱细胞的图像轰击不同质粒结构。 （一）和（D），洋葱细胞轰炸35S：：GFP的。绿色荧光蛋白扩散通过了细胞质和细胞核。 （二）及（E），洋葱细胞35S等于摩尔混合轰炸：SEU - pSPYNE（SEU的YFP的N -端片段融合）和35S：：LUH pSPYCE（LUH融合的C -末端片段）质粒DNA。 SEU - pSPYNE LUH - pSPYCE之间的相互作用是由荧光的细胞核。 （三）及（F），阴性对照显示洋葱细胞与载体pUC19 SPYNE LUH - pSPYCE plasmi混合轰炸DS。没有检测到荧光信号。

<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> Mol Cell. 9 (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> Plant Physiol. 147 (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> Plant J. 40 (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> J Vis Exp. , (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> Plant Physiol. 146 (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> Plant Physiol. 150 (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6（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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<td>Yellow onion</td>
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<td>Any supermarket</td>
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<td>Helios Gene Gun</td>
<td>&nbsp;</td>
<td>BioRad</td>
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<td>Fluorescent microscope with appropriate filters for GFP or YFP</td>
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bimolecular fluorescence complementation (bifc) assay for protein-protein interaction in onion cells using the helios gene gun

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。

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

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

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 ...

bimolecular-fluorescence-complementation-bifc-assay-for-protein

Research

JoVE Journal

Biology

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

19.5K 次观看.  University of Maryland. 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

视频: 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

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

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

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

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

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

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

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

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

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

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

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

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

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate 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

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

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

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

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) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

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 ...