私たちは、原稿と一般的なラボの援助を読み取るためのVi Nguyenさんに感謝。

1。 アグロバクテリウム培養液の調製<ol><li> アグロバクテリウム菌株GV3101は、以前にゲートウェイの互換性のあるBiFCベクトルpEarleyGate201 - YCとpEarleyGate202 - YN、融合GOI構築物を含む各で形質転換。 </li><li> BiFC融合タンパク質の5 mlのYEB（5G L -1の牛肉エキス、1G L -1の酵母エキス、5G L -1ペプトン、5G L -1スクロース、2mMのMgSO 4を用い 、pH7.2）で培養に接種することでアグロバクテリウムを形質転換コンストラクト適切な抗生物質選択。 28で一晩培養して成長℃〜200 rpmで振盪。 </li><li>滅菌済み1.5 mL遠心管に培養液1 mlを取り外します。 </li><li>室温で10分間、1,000 gでペレット細胞は、上清を捨てる。 </li><li>浸透培地1ミリリットル（5グラムL -1 D -グルコース、50mMのMES（シグマ）、2mMののNa 3 PO 4、0.1mMのアセトシリンゴン）11を追加することでペレットを洗浄します。ピペッティングでペレットを再懸濁します、10分1,000 gで、再度、細胞をペレット化する。 </li><li>洗浄工程を2回繰り返します。 </li><li> 0.5mlの浸潤のメディアでペレットを再懸濁する。 </li><li> 600 nmで吸光度を測定し、1.2に1.0にOD 600の最終調整。 </li><li>新しい1.5 mlチューブに各構成要素のアグロバクテリウム懸濁液を同量、10秒間ボルテックスアリコート。単一の浸潤のため、各構築物の100μLは、十分以上のものです。 </li><li> アグロバクテリウムの混合物は、現在浸透のための準備ができています。 </li></ol> 2。浸潤<ol start="11"><li> N. benthamiana植物は、16時間明＆8時間暗のサイクルで、21℃で栽培されている。 5〜6週齢の植物は、浸透のために使用する必要があります。 </li><li>気孔が完全に開くことができますinfiltratring前に1時間の白色光の下で成長室と場所から植物を取り出します。 </li><li> 1 mlのスリップ先端ツベルクリン注射器の針なしで再懸濁したアグロバクテリウム混合物を描く。 </li><li>葉の裏面に対して、シリンジの先端を置き、静かに直接指一本で葉の表側をサポートしながら、プランジャーを押し下げる。それはmesophyllar空気のスペースを埋めるように液体が葉を通して拡散する。 </li><li>将来の識別用マーカーペンで浸透した領域にラベルを付けます。 </li><li>異なる構造を浸潤する場合は、手袋を変更したり、浸潤の間に70％エタノールでそれらをきれいにするのどちらかを確認してください。可能であれば、交差汚染を防ぐために、異なるサンプル間に1つの中肋のスペースを残す。 </li><li>正常な成長条件下で成長キャビネットに植物を置きます。 </li></ol> 3。再構成されたYFP信号の観測<ol start="18"><li>浸透ゾーン内の葉組織の税5mm x 5mmのセグメント</li><li>水の中で、ガラスの顕微鏡スライド上に、カバースリップでサンプルをカバーし、共焦点または蛍光顕微鏡を用いて相互作用を調べ、サンプルをマウントします。 </li><li>時間の経過とともにいくつかの異なる場所を表示する蛍光融合タンパク質をもたらすことができます発現/人身売買以上のように式は最大5日後に浸潤の時間から24時間ごとに監視する必要があります。 </li></ol> 4。代表的な結果： BiFCアッセイによりタンパク質 - タンパク質相互作用の例を図1に示されています。 AtTHP1とAtSAC3Aは酵母TREX - 2複合体のシロイヌナズナホモログの成分であると考えられている。彼らはnuclearporin AtNUP1と相互作用し、mRNAの輸出10を容易にすることができます。 AtNUP1がpEarlyGate202 - YN中にクローニングされている間AtTHP1とAtSAC3AはpEarlyGate201 - YCにクローニングした。構造は続いてGV3101に形質転換した。浸透のために三つの構成は、3通りの組み合わせ（AtNUP1 + AtSAC3A; AtTHP1 + AtSAC3A AtTHP1 + AtNUP1）と対にされた。再構成されたYFPのシグナルは48時間浸透後LCSM下で観察した。信号は、表皮細胞で観察し、これらのタンパク質のサブ細胞内局在と一致している核周縁部や核プラズマ、に局在していた。ネガティブコントロールは、YFPのシグナルを示していない。 <img src="/files/ftp_upload/3473/3473fig1.jpg" alt="図1" /> 図1 シロイヌナズナ TREX - 2 mRNAの輸出複雑なコンポーネントの相互作用。N. benthamiana葉、GOIの構造との共同変換は、ライカTCS SP2 confocol顕微鏡を使用して、浸潤後48〜72時間をイメージした、pEarleyGate201 - YCとpEarleyGate202 - YN（として示される）に融合。画像は、マージされた共焦点YFPと表皮Nの明視野像として表示されますbenthamiana葉細胞

<ol>
	<li>Ohad, N., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+bimolecular+fluorescence+complementation+(BiFC)+to+assay+protein-protein+interaction+in+plants.">Utilizing bimolecular fluorescence complementation (BiFC) to assay protein-protein interaction in plants.</a> Methods in molecular biology. 655, 347-358 (2010).</li><li>Fang, Y., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiFC+imaging+assay+for+plant+protein-protein+interactions.">BiFC imaging assay for plant protein-protein interactions.</a> Cold Spring Harbor protocols. 2, (2010).</li><li>Schutze, K., Harter, K., Chaban, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+to+study+protein-protein+interactions+in+living+plant+cells.">Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells.</a> Methods in molecular biology. 479, 189-202 (2009).</li><li>Hiatt, 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=Bimolecular+fluorescence+complementation+(BiFC)+analysis+of+protein+interactions+in+Caenorhabditis+elegans.">Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans.</a> Methods. 45, 185-191 (2008).</li><li>Barnard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+implementation+of+split-GFP-based+bimolecular+fluorescence+complementation+(BiFC)+assays+in+yeast.">Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast.</a> Biochemical Society Transactions. 36, 479-482 (2008).</li><li>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=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nature. 1, 1278-1286 (2006).</li><li>Hu, C. D., Grinberg, A. V., 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+protein+interactions+in+living+cells+using+bimolecular+fluorescence+complementation+(BiFC)+analysis.">Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis.</a> Current protocols in cell biology. 21, 3-3 (2006).</li><li>Earley, K. W. <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+vectors+for+plant+functional+genomics+and+proteomics.">Gateway-compatible vectors for plant functional genomics and proteomics.</a> The Plant journal : for cell and molecular biology. 45, 616-629 (2006).</li><li>Karimi, M., Inze, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY vectors for Agrobacterium-mediated plant transformation.</a> Trends in Plant Science. 7, 193-195 (2002).</li><li>Lu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+homolog+of+the+yeast+TREX-2+mRNA+export+complex:+components+and+anchoring+nucleoporin.">Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin.</a> The Plant journal : for cell and molecular biology. 61, 259-270 (2010).</li><li>Sparkes, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transient+expression+of+fluorescent+fusion+proteins+in+tobacco+plants+and+generation+of+stably+transformed+plants.">transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.</a> Nature protocols. 1, 2019-2025 (2006).</li><li>Rackham, O., Brown, C. 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+RNA-protein+interactions+in+living+cells:+FMRP+and+IMP1+interact+on+mRNAs.">Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs.</a> The EMBO journal. 23, 3346-3355 (2004).</li><li>Demidov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+complementation+of+split+fluorescent+protein+triggered+by+DNA+hybridization.">Fast complementation of split fluorescent protein triggered by DNA hybridization.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 2052-2056 (2006).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <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+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nature. 3, 1693-1702 (2008).</li></ol>

利害の衝突は宣言されません。

BiFCアッセイはタンパク質相互作用を研究するための強力なツールです。従来のY2Hアッセイとは異なり、BiFCだけでなく、タンパク質 - タンパク質相互作用を可視化できるだけでなく、サブ細胞内局在などのタンパク質複合体の詳細な情報を提供します。また、2つの候補タンパク質が第三のパートナーが十分に近づけることができる限り、間接的な相互作用を検出することが可能です。あらゆ...

<table border='1'>
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma</td>
			<td>M2933</td>
		</tr>
		<tr>
			<td>1 mL slip-tip tuberculin syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
	</tbody>
</table>

detection of protein interactions in plant using a gateway compatible bimolecular fluorescence complementation (bifc) system

我々は、 生体内の 2つのタンパク質間相互作用をテストするBiFC技術を開発しました。これは二つの非重複フラグメントに分割することにより、黄色蛍光タンパク質（YFPを）実現されます。各断片は、目的の遺伝子にインフレームでクローニングされる。これらのコンストラクトは、融合タンパク質の輸送の発現を可能にする、 アグロバクテリウム媒介形質転換によりタバコbenthamianaに同時形質転換することができる。検死のタンパク質が1-7をやり取りするときにYFPシグナルの再構成にのみ発生します。タンパク質 - タンパク質相互作用をテストおよび検証するために、BiFCは酵母ツーハイブリッド（Y2H）アッセイと一緒に使用することができます。これは、Y2Hで見落とされることができる間接的な相互作用を検出する場合があります。 Gatewayテクノロジは、できるだけ8,9限り多くの発現および機能解析システムにシャトルに興味のある遺伝子（GOI）の研究者を可能にする普遍的なプラットフォームです。方向とリーディングフレームの両方が表現対応のクローンを作るために制限酵素やライゲーションを使用せずに維持することができます。結果として、一つは実験全体で一貫した結果を確保するために、すべての再シーケンシングのステップをなくすことができます。我々はBiFCとY2H両方アッセイ10を実行するために使いやすいツールを研究者に提供するゲートウェイ互換BiFCとY2H一連のベクトルを作成している。ここで、我々は、Nのタンパク質-タンパク質相互作用をテストするために私達のBiFCシステムを使用してのしやすさを示すbenthamiana植物 。

Watch this Scientific Journal Video about Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System at JoVE.com

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

我々は、植物におけるタンパク質間相互作用をテストする手法を開発した。黄色蛍光タンパク質（YFP）がつの非オーバーラップフラグメントに分割されます。各断片は融合タンパク質の発現を可能にする、ゲートウェイのシステムを経由して目的の遺伝子にインフレームでクローニングされる。検死のタンパク質が相互作用するときにYFPシグナルの再構成にのみ発生します。

ゲートウェイ互換二分子蛍光相補（BiFC）システムを使用してプラントにおけるタンパク質相互作用の検出

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

detection-protein-interactions-plant-using-gateway-compatible

Research

JoVE Journal

Biology

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

25.2K ビュー.  University of Western Ontario. 我々は、植物におけるタンパク質間相互作用をテストする手法を開発した。黄色蛍光タンパク質（YFP）がつの非オーバーラップフラグメントに分割されます。各断片は融合タンパク質の発現を可能にする、ゲートウェイのシステムを経由して目的の遺伝子にインフレームでクローニングされる。検死のタンパク質が相互作用するときにYFPシグナルの再構?...

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

生物学

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

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

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

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

ヘリオス遺伝子銃を用いたタマネギの細胞における蛋白質 - 蛋白質相互作用のための二分子蛍光相補（BiFC）アッセイ

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

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

マイクロパターン化表面でのタンパク質間相互作用の in vivo検出

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

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.

二分子蛍光相補

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

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

に基づいて、タンパク質 - タンパク質相互作用の同定のためのプロトコル<sup&gt; 15</sup&gt; Nの代謝標識、免疫沈降法、定量的質量分析法と親和性調節

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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.

二分子蛍光相補のフローサイトメトリー分析：タンパク質 - タンパク質相互作用を研究するためのハイスループット定量法

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.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

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.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

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

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

示差走査蛍光光度を用いたタンパク質 - リガンド相互作用の定量

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

蛍光プロテアーゼ保護を使用して膜タンパク質のトポロジの決定（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...