我々はGPI - GFPについては、hCD71 - GFPコンストラクト、及びダニエルLegler、コンスタンツ、スイスの大学のために、彼女の親切な助け、カタリーナシュトループ、ジュネーブ、スイスの大学のためQuentinaビーティ、リンツ、オーストリアの大学を、感謝したいと思います- DAFの構築。と科学研究のためのオーストリア連邦省のGEN - AUのプロジェクト、この作品は、オーストリア科学基金（プロジェクトY250 - B03 FWF）によってサポートされていました。

マイクロコンタクトプリンティング： <ol><li>メスを用いてPDMSスタンプの微細のフィールドをカット</li><li>エタノール（100％）と蒸留水でそれをフラッシュすることにより微細を含むフィールドを洗ってください。 </li><li>窒素またはアルゴンガスでPDMSスタンプを乾かします。 </li><li>ピペットを50μlBSA - Cy5と仕事のソリューション（0.67 mg / ml）をPDMSスタンプ上に全体の微細フィールドが溶液で覆われるように。室温で30分間インキュベートする。 </li><li>リン酸でそれをフラッシュすることにより微細フィールドを洗うには、生理食塩水（PBS）と蒸留水をバッファ。 </li><li>窒素またはアルゴンガスでPDMSを乾かします。 </li><li>個のエポキシコーティングされたカバースリップの途中に自重でPDMSスタンプを配置し、ペトリ皿の中で、室温で30分間インキュベートする。 </li><li>カバーの裏面にPDMSスタンプの位置にラベルを付け、ピンセットを用い、スライドからスタンプを取り除く。 </li><li>カバースリップ上にマイクロコンタクト印刷されたフィールドを介してセキュアシールのハイブリダイゼーションチャンバーを貼る。ラベルは、フィールドの中心をローカライズするのに役立ちます。 </li><li>ピペット60μlのストレプトアビジンの作業溶液（50μg/ ml）を反応室に、室温で60分間サンプルをインキュベートする。 </li><li>チャンバーの一方のポートにバッファを追加し、ポンプで2番目のポートで同時にそれを除去することによって1mlのPBSでサンプルを洗浄してください。 </li><li>ピペット60μlのビオチン化抗体の作業溶液（10μgの/ 0.1％ツイーン20を添加したPBS中、&quot;PBST&quot;）室温で60分間反応チャンバとインキュベートに。 </li><li>チャンバーの一方のポートにバッファを追加し、ポンプで2番目のポートで再びそれを削除することによって、1 mlのPBSTと続いて1mlのPBSでサンプルを洗浄してください。 </li><li>細胞が播種するための準備が整うまでは、暗所、室温でPBSでマイクロパターン表面を保管してください。 </li></ol>マイクロパターン表面への細胞のインキュベーション： <ol><li> 3cmの組織培養プレートで50％コンフルエントに接着細胞を成長させる。 </li><li>エチレンジアミン四酢酸（EDTA）溶液で関心の餌と獲物タンパク質を発現する細胞を剥​​離し、1,000 rpmで5分遠心してください。 </li><li>上清を捨て、応じて増殖培地中で細胞ペレットを溶解する。 </li><li> 1,000 rpmで5分間遠心操作する。 </li><li>上清を捨て、適切な増殖培地中で細胞ペレットを溶解する。 </li><li>交換細胞懸濁液60μlのでマイクロパターンのカバースリップ上で反応チャンバーからPBS。 </li><li>蒸留水で滅菌パッドを浸漬し、乾燥した実行サンプルを防ぐために、ペトリ皿にそれを置くことによって加湿チャンバーを作成します。 </li><li> 37泊以上の細胞をインキュベートしますマイクロパターン表面上に、5％CO 2雰囲気でC。 </li><li>のCa 2 +およびMg 2 +を含むハンクスのバッファ塩溶液による顕微鏡、交換培地上の細胞を分析する前に。 </li></ol>顕微鏡： <ol><li>カバースリップを適切なマウントで配置され、細胞の形態は、白色光下でチェックされます。 </li><li> BSA - Cy5のパターンの品質は、647 nmで励起によってチェックされます。 </li><li>蛍光preyタンパク質（GFPまたはYFP）の微細パターンは、全反射（TIR）励起の下で488または514 nmで記録されます。 </li></ol>代表的な結果： マイクロパターン表面上に成長させた細胞中の標的タンパク質間の相互作用がある場合は、獲物が餌の再分配に従います。結果として得られる微細はpreyタンパク質の蛍光標識によって可視化することができる。重要なのは得られるコントラストは、相互作用の強さを直接測定（図1参照）を提供しています。したがって、2つのタンパク質の相互作用の簡易評価が可能となる - さらに、測定主要なデータを処理するために必要なし。 <img src="/files/ftp_upload/1969/1969fig1.jpg" alt="図1" /> 図1。異なる相互作用強度で生細胞の原形質膜中の餌の転位。 CD59抗体microbiochipsでTIRのCD71抗体で（A）CD71 - GFPをトランスフェクションしたT24細胞の画像、（B）CD4およびCD4抗体で細胞質YFPと（C）GPI - GFP - DAFが表示されます。データは、（）強いの特性がない（b）または弱い相互作用（c）があります。 （A）（Weghuber ら 、印刷中）、（B）（Schwarzenbacher ら 、2008）からから再生される。

<ol>
	<li>Brameshuber, M., Schwarzenbacher, M., Kaltenbrunner, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schuetz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reply+to+"Uncoupling+diffusion+and+binding+in+FRAP+experiments.">Reply to "Uncoupling diffusion and binding in FRAP experiments.</a> Nat. Methods. 6, 183-184 (2009).</li><li>Kim, P. W., Sun, Z. J., Blacklow, S. C., Wagner, G., Eck, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Zinc+Clasp+Structure+Tethers+Lck+to+T+Cell+Coreceptors+CD4+and+CD8.">A Zinc Clasp Structure Tethers Lck to T Cell Coreceptors CD4 and CD8.</a> Science. 301, 1725-1728 (2003).</li><li>Ramm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+screening:+a+technology+in+transition.">Image-based screening: a technology in transition.</a> Curr. Opin. Biotechnol. 16, 41-48 (2005).</li><li>Schwarzenbacher, M., Kaltenbrunner, M., Brameshuber, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+for+quantitative+analysis+of+protein-protein+interactions+in+living+cells.">Micropatterning for quantitative analysis of protein-protein interactions in living cells.</a> Nat. Methods. 5, 1053-1060 (2008).</li><li>Weghuber, J., Brameshuber, M., Sunzenauer, S., Lehner, M., Paar, C., Haselbruebler, T., Schwarzenbacher, M., Kaltenbrunner, M., Paster, W., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein-protein+interactions+in+the+live+cell+plasma+membrane+by+quantifying+prey+redistribution+upon+bait+micropatterning.">Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.</a> Methods in Enzymology. , .</li></ol>

ビデオでは、可視化研究室のホームページから取らオリンパス、、ブラウン大学によって提供される（Kim ら 、2003）から再生された画像が含まれています。

付属のビデオでは、生きた細胞の血漿膜（。。Brameshuber ら 、2009;。。Weghuber ら 、 印刷中 Schwarzenbacher ら 、2008）におけるタンパク質間相互作用を検出する方法を示しています。原則として、いかなるTIRFベースの顕微鏡のプラットフォームは、読み出しシステムとして使用することができます。高感度は（単一分子の検出用など）が必要なときにのみ、高度な顕微鏡が必要になります。最良の結果を達成するための準備処理中に、次の重要な点は特別な注意が必要です。 <ol class="lroman"><li>暗条件下でのストアでは、Cy5標識ストック溶液蛍光体の漂白を避けるとマイクロコンタクトプリンティングの前にたてBSA - Cy5と作業溶液を調製する。 </li><li>ピペットの先端でPDMSにマイクロパターンを掻くと蛍光体の漂白を避けるために暗い条件下でサンプルをインキュベートしないように注意してください。 </li><li>ペトリ皿にガラスのスライドの固着を防ぐために、滅菌パッドの上にカバースリップを置く。 PDMSスタンプは、スライドガラスに付着したら、それを移動しないでください。 </li><li>反応室内の空気の泡を避け、蛍光団の漂白を避けるために暗い条件下でサンプルをインキュベートする。 </li><li>塩の結晶の形成を避けるために2分以上のサンプルを乾燥させないでください。 </li><li>細胞の剥離用としてトリプシンを使用しないでください、それを切断するには、これまでに発現する膜タンパク質の細胞外ドメインなります。トリプシンを避けることがすぐに表面上の細胞の付着後に微細パターンを形成するために低離職率を持つタンパク質の参考になります。 </li><li>顕微鏡でインキュベートした細胞の数を制御し、細胞がより30-50％コンフルエントに達していないことを確認してください。過剰な細胞が原因で抗体でコーティングされた表面上の細胞の高速アタッチメントにすぐに削除されていることを確認します。 </li><li>高品質のBSA - Cy5のパターンを持つ唯一のスライドは、さらなる分析のために使用することができます。 </li><li>蛍光preyタンパク質の安定した発現が原因で、スキャンごとに分析することができるセルの数が多いのが望ましい。 </li><li>十分な全反射の調整は、細胞質背景に起因するパターンを可視化することが不可欠である。 </li></ol>マイクロパターニング技術は、タンパク質 - タンパク質相互作用の分析のためのいくつかの可能性を提供しています。まず、ローカル、空間分解タンパク質間相互作用の定量化と一緒にも弱いまたは間接的な相互作用の検出は、偽陽性または陰性の高い数を与えることの欠点なしで可能です。第二にそれは2 - ハイブリッドスクリーンのような生化学的なアプローチによって達成することは困難である生きた細胞の血漿膜にタンパク質間相互作用を分析する研究が可能になります。第三のアプローチは、温度などの環境変化、​​異なるタンパク質または他の分子や翻訳後修飾の存在によって変調される餌と被食者相互作用の検出が可能になります。したがって、このアッセイは、生細胞のコンテキスト内の指定された相互作用のペアのスクリーニング変調が可能になります。さらに、捕獲リガンドまたは一価のリガンドの使用の面密度を低減することにより、静止状態の解析が可能となる。適切なスキャンのプラットフォームが使用されている場合、最終的には、分析セルの数は、薬物スクリーニングのための製薬企業の高スループット要求（ラム、2005）と一致するのに十分高いです。

in-vivo detection of protein-protein interactions on micro-patterned surfaces

分子の相互作用のネットワークを解明<em&gt;生体内</em&gt;細胞機能と代謝の制御機構を理解する鍵である。細胞内の分子間相互作用に対処するための方法論的な多数のオプションが開発されたが、これらのメソッドのほとんどは、ほとんど定量的ではなく、間接的、従ってであることに苦しんでされています。逆に、少数のハイエンド定量的アプローチは、しかし、高いスループットに拡張することが困難である、導入されました。定量的な情報を抽出する可能性が高いスループット能力を結合するには、我々は最近、タンパク質間相互作用を識別するための新しいコンセプトを（Schwarzenbacher開発<em&gt;ら</em&gt;。、2008）。ここで、我々はデザインと蛍光標識タンパク質（&quot;獲物&quot;）と膜タンパク質（&quot;餌&quot;）との間の相互作用を分析することを可能にするこのシステムの構築のための詳細なプロトコルを記述する<em&gt;生体内</em&gt;。細胞は、餌exoplasmicドメインに対する抗体で機能化マイクロパターン表面上にメッキされています。餌 - 獲物相互作用は、蛍光獲物の再分配を経由してアッセイされる。方法は、スクリーニングツールとしてそれが適用されること、ダウン、単一分子のレベルまで高感度、弱い相互作用を検出する能力、および高いスループット能力によって特徴づけられる。

Watch this Scientific Journal Video about In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces at JoVE.com

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

このビデオでは、マイクロパターン化表面を使用することにより、タンパク質 - タンパク質相互作用のその後の分析と実験を示しています。アプローチは、生きた細胞でタンパク質間相互作用を検出する可能性を提供し、定量的な情報を抽出する可能性が高いスループット能力を兼ね備えています。

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 ...

in-vivo-detection-protein-protein-interactions-on-micro-patterned

Research

JoVE Journal

Biology

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

10.7K ビュー.  Johannes Kepler Universitat Linz. このビデオでは、マイクロパターン化表面を使用することにより、タンパク質 - タンパク質相互作用のその後の分析と実験を示しています。アプローチは、生きた細胞でタンパク質間相互作用を検出する可能性を提供し、定量的な情報を抽出する可能性が高いスループット能力を兼ね備えています。

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

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

生物学

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

イメージングタンパク質間相互作用 in vivoで</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

スペクトル的に解決された二光子顕微鏡を用いたGタンパク質共役受容体相互作用の in vivo定量で

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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 ...

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 ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

タンパク質 - タンパク質相互作用の阻害剤の開発を通じてREPLACE：アプリケーションの設計と開発非ATP競合CDK阻害剤に

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

シロイヌナズナタンパク質-DNA相互作用の同定のための免疫沈降アッセイクロマチン<em&gt;インビボ</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

オートファジー研究でタンパク質間相互作用の研究

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...