著者らは、放射性標識されたウイルスを調製するための医薬バイオテクノロジーと分子生物学の部門、特にモニーク·デ·Pelsmackerのスタッフに感謝します。私たちは、エレン·メルクスとその興味深い発言や議論のためにHadewych Halewyckに、ラボで彼の助けのためのヘリット·デ·Bleeserに感謝しています。この作品は、財政的にブリュッセル自由大学（OZR1807）、フォンvoor Wetenschappelijk Onderzoe​​kブラーンデレン（G.0168.10N）と世界保健機関（TSA 200410791）のOZRの助成金によって支えられている。リセSchotteはフォンvoor Wetenschappelijk Onderzoe​​kブラーンデレン（FWO）の博士号を取得する前の仲間です。

原則（）とメソッドの概要（B）は図1に示されている。 1。バッファーの調製<ol><li>水の中でリン酸二水素ナトリウム（50 mm）と塩化ナトリウム（300 mM）を溶解することによって結合/洗浄バッファーを準備し、pHを8.0に調整します。さらに、メチオニンツイーン20（0.01％（M / V）の最終濃度）、（2％（M / V）の最終濃度）とアルブミン（0.1％（M / V）の最終濃度）を追加し、必要なボリュームに調整します。 </li></ol> 2。磁性ビーズの調製取扱説明書に従って、磁気ビーズを準備します。簡潔に： <ol><li>渦を用いた磁気ビーズを再懸濁します。 </li><li>転送は、各サンプルの、チューブに再懸濁した磁性ビーズ（40 mg / ml）を10μlの。 </li><li>上清が透明になるまで、磁石を用いた磁気ビーズを収集する（+ / - 30秒）し、上清を注意深く除去する。 </li><li>洗う磁気ビーズを2回：結合/洗浄バッファー150μlでビーズを再懸濁します。磁気ビーズ上清が透明になるまで磁石を用いたチューブの一方の側に移行し、慎重に上清を除去します。 </li><li>ビーズ（10 mg / ml）を懸濁するため、各サンプルについて、結合/洗浄バッファー40μlを使用しています。 </li></ol> 3。親和性捕捉アッセイ注：（宇宙の）バックグラウンド放射能（0％放射能）を測定するには、no放射標識されたウイルスは対照試料1に追加されていません。代わりに、同じボリューム結合/洗浄バッファーが追加されます。 注：100％の放射能が、すべてのコンポーネントがnanobodyのを除いて追加されるように制御サンプル2で定義されています。代わりに、同じボリューム結合/洗浄バッファーが追加されます。 <ol><li> 96ウェルマイクロタイタープレートに80μlに結合/洗浄バッファーで希釈した（β-放射）ポリオウイルスセービン株の種類1〜9、標識された35 Sの2000 CPMをもたらします。 </li><li> 10＆mを追加するU;ウェルにnanobodyの希釈lの。 </li><li>シェーカーを使用して約10秒間混ぜる。 </li><li>サンプルは室温で1時間インキュベートすることができます。 </li><li>洗浄した磁気ビーズ懸濁液40μlを追加し、連続的に振盪し、室温で10分間インキュベートします。 </li><li>ビーズとマグネットを使用して上清を分離し、チューブにクリアし、上清を移す。 </li></ol> 4。放射能の測定<ol><li>カウントフラスコに、ステップ3.6からの上清50μlを転送します。 </li><li>シンチレーション液と混合の3ミリリットルを追加します。 β-シンチレーションカウンターで放射能を測定します。 </li></ol> 5。ビーズのリサイクル<ol><li> 2分間500 xgで、または透明な上清が得られるまで、チューブと遠心分離機で使用される磁気ビーズを収集します。 </li><li>上清を除去し、6ミリリットル、0.5 M NaOHでビーズを再懸濁します。 </li><li> multiplに懸濁液を移す電子管と5分間超音波浴中でインキュベートします。 </li><li>磁気ビーズを収集し、上清を除去するために磁石を使用しています。 </li><li>各チューブに1 mLの2％SDS-ソリューションを追加し、ボルテックスを用いて再懸濁します。 5分間沸騰させる。 </li><li>ビーズを収集し、上清を除去します。 1ミリリットルの0.2 M EDTA（pH = 7）でビーズを再懸濁します。 </li><li> 5分間超音波浴中でチューブをインキュベートし、もう一度、上清を除去します。 </li><li>各チューブに1 mlの水を追加して、ビーズを懸濁します。ビーズを収集し、上清を除去します。二回の洗浄ステップを繰り返します。 </li><li>上清を除去し、1mlの10mMのCoCl 2溶液中でビーズを再懸濁します。 10分間シェーカー上に置く。 </li><li>ビーズを収集するために磁石を使用して上清を除去し、結合/洗浄バッファー1 mlに再懸濁します。 </li><li>上清を除去し、20％（v / v）エタノール1 mlを用いて2回ビーズを洗浄</li><li> 20％の元の体積（v / v）の他にビーズを再懸濁し40 mg / mlの濃度で再生ビーズを得るためにhanol。 </li></ol> 6。結果の解釈<ol><li>ない放射性標識ウイルスを添加しなかった対照試料1は、バックグラウンド放射能を修正するために、0％の放射能値を設定するために使用されます。 nanobodyのが含まれており、その結果、すべての放射標識されたウイルスは上清に残っている、されないコントロールサンプル2は、100％の放射能値として設定されています。 </li><li> nanobodyので放射性標識ウイルスの - 上清中の放射能の割合（=％）が全体の降水量（％= 100）の測定値です。 </li></ol> 7。代表的な結果この親和性捕捉アッセイの代表的な結果は、 図2、図3と図4に示されています。 図2では、PVSS38C、ポリオウイルスのためにnanobodyの、特定の、の親和性が示されています。 PVSS38Cの親和性はpoliovの3つの異なる抗原に対してテストされていますirus：ネイティブ抗原（N-抗原）、加熱抗原（H抗原）と14Sサブユニット。 N-抗原は感染性である無傷のウイルスです。ウイルスは加熱（56時20分°C）または固体表面（未発表データ）、ウイルスRNAの（部分的）損失とキャプシドタンパク質VP4の結果カプシド変更のコンフォメーション、空に接続されている場合キャプシドは、その後、H抗原と呼ばれるが形成される。 14Sサブユニットは、アセンブリの中間体である。これらの抗原は、免疫後10抗体の異なるセットで認識されるため、異なるエピトープと抗原部位を持ってしています。図では上清中に見つかった放射能の割合はnanobodyの濃度の関数として表されます。それは放射性標識14Sサブユニットを含むサンプルの上清中の放射能はnanobodyのPVSS38C濃度の増加とともに減少することがわかる。これは、14Sサブユニットの消費ポリオウイルス量の増加のように変換することができますnanobodyのPVSS38Cおよび間接的に磁気ビーズに接続されています。キャプチャ価（放射性標識ウイルスの50％を捕捉するために必要なnanobodyの数=濃度）がグラフィカルに0.099 nMのように計算することができます。ポリオウイルスのN抗原とH抗原性フォームのPVSS38Cの親和性が観察されていません。これらのデータからそれがPVSS38Cは、ポリオウイルスの二つの他の抗原フォーム上の排他的に14Sサブユニット上に存在していないエピトープと相互作用していると解釈されます。 上清からの放射能の消失だけで、その抗原に対するnanobodyの特定の親和性によるものであることを示すために、同一のアッセイは、スルホロバスsulfataricusのlrpB転写制御因子に対して生成されたnanobodyの、NB1で行われ、との相互作用を持たないことが知られていた任意のポリオウイルス抗原。このアッセイの結果を図3に示します。すべての放射性標識ウイルスのない反応がないことを示す上清に残っているポリオウイルスの3抗原の形態に対してNB1。 結果の再現性は、別の日に一つ与えられたnanobodyの（すなわち、PVSP29F）の8回の合計で実験を繰り返すことで、異なる人によってテストされています。結果を図4に示されています。上清中の放射能の割合の平均値は、各nanobodyの濃度を算出し、その標準偏差に対応して表されます。 <img alt="図1。" src="/files/ftp_upload/3937/3937fig1.jpg" /> 図1。アッセイの原理（A）の概要と方法（B）。 A. Hisタグ特異的に特定のポリオウイルスの抗原を認識ナノボディは、コバルト被覆磁気ビーズと対話することができますし、放射標識されたウイルスを沈殿させます。 B. Hisタグナノボディは、放射性標識したポリオウイルスを添加し、インキュベートする。コバルト被覆磁気ビーズです。追加され、バインド/非結合抗原の磁気分離が実行されます。上清の放射能が測定され、キャプチャされた抗原の量を導出することができます。 <img alt="図2" src="/files/ftp_upload/3937/3937fig2.jpg" /> 図2。 nanobodyのPVSS38Cによって異なるポリオウイルス抗原の磁気ビーズをアフィニティーキャプチャー 。上清中に見つかった放射能の割合はPVSS38Cの濃度の関数として表されます。 14S用濃度 - 反応関係は14Sに排他的に存在するエピトープとPVSS38Cの相互作用を示す、見つけることができます。非常に高濃度で無視非特異的取り込みが注目されているものの、N-、また、H-抗原との有意な相互作用が検出できません。 <img alt="図3" src="/files/ftp_upload/3937/3937fig3.jpg" /> 図3。 nanobodyのNB1によって異なるポリオウイルス抗原の磁気ビーズの親和性のキャプチャ </stronG&gt;。上清中に見つかった放射能の割合は、NB1の濃度の関数として表されます。 NB1は、任意のポリオウイルス抗原またはサブユニットとの相互作用を持たないことが知られています。放射能の100％が上清中に検出されたので、どんなウイルスが非特異的にNB1にバインドされませんでした。 図2中の放射能の消失は、したがって、その抗原に対するnanobodyの特定の親和性のみによるものである。 <img alt="図4。" src="/files/ftp_upload/3937/3937fig4.jpg" /> 図4。アッセイの再現性 。上清中の放射能の割合の平均値はnanobodyの濃度の関数として表されます。実験は、異なる日に、別の人による8回繰り返した。標準偏差は、縦棒として表されます。

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我々は、開示することは何もありません。

プロトコルでは、nanobodyのと相互作用する抗原の放射能を上清からの放射標識されたウイルスの損失として定義されています。したがって、沈殿した放射能（= 1​​00 - ％）の量は、（磁気ビーズに結合した）上清中の放射能（=％）で間接的な方法で推定することができます。その一方で、それは500 mMのイミダゾールを用いた磁気ビーズから免疫複合体を溶出することにより直接的な方法で?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong試薬の&gt;名前</strong</td&gt;<td<strong&gt;会社概要</strong</td&gt;<td<strong&gt;カタログ番号</strong</td&gt;<td<strong&gt;コメント</strong</td&gt;</tr&gt;<tr&gt;<td&gt;ダイナHisタグの単離とプルダウン</td&gt;<td&gt;インビトロジェン</td&gt;<td&gt; 101.03D</td&gt;<td&gt;磁気ビーズ</td&gt;</tr&gt;<tr&gt;<td&gt; Optiphase &#39;HiSafe 2&#39;</td&gt;<td&gt;パーキンエルマー</td&gt;<td&gt; 1200 - 436</td&gt;<td&gt;シンチレーション流体</td&gt;</tr&gt;</tbody&gt;</table

a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example

この記事では、単純な、定量、液相の親和性捕捉アッセイが提示されています。一つのタンパク質をタグ付けすることができます提供され、別のタンパク質が標識され、このメソッドは、タンパク質 - タンパク質相互作用の調査のために実装することができます。これは、コバルト被覆磁性ビーズによるタグ付きタンパク質の認識に、タグ付きタンパク質と標識された第2の特異的なタンパク質間の相互作用で一方一方に基づいています。最初に、ラベルとタグ融合タンパク質を混合し、室温でインキュベートされています。タグを認識した磁性ビーズは、追加され、標識タンパク質の結合画分は、磁石を使用して非結合画分から分離されています。キャプチャされた標識タンパク質の量は、非結合画分に残った標識タンパク質の信号を測定することにより、間接的な方法で決定することができます。コンフォメーションの変換に敏感なタンパク質が尻である場合に記載の液相親和性アッセイは、非常に便利です。ayed。アッセイの開発とアプリケーションがポリオウイルスとナノボディ1を認識してポリオウイルスの間の相互作用を実証しています。ポリオウイルスは、固体表面（未発表データ）に接続されたコンフォメーションの変換2〜敏感であるため、ELISAの使用が制限されており、液相ベースのシステムは、したがって、優先されるべきである。しばしばpolioresearch 3,4で使用されている液相ベースのシステムの例としては、マイクロタンパク質免疫沈降テスト5です。このテストは、その適用性を実証しているにもかかわらず、それが6,7ナノボディで不在であるFcの構造を必要とします。ただし、別の機会として、これらの興味深いかつ安定した単一ドメイン抗体8は簡単に別のタグを使用して設計することができます。広く（彼の）6-tagはそのような自分の順番で簡単に磁気ビーズ上にコーティングすることができ、ニッケルやコバルトなどの二価イオンに対する親和性を示しています使用されます。したがって、我々はこの単純な定量を開発しましたコバルト被覆磁性ビーズに基づく親和性捕捉アッセイ。ポリオウイルスは、ナノボディで妨害さの相互作用を有効にして定量的な検出が可能にする35 Sで標識した。メソッドは、実行が容易で、さらに効果的に再生磁気ビーズの可能性によってサポートされている、低コストで確立できます。

Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

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

この記事では、単純な、定量、液相の親和性捕捉アッセイが提示されています。それは一方では、磁気ビーズとタグ融合タンパク質（例えば、ナノボディ）との間の相互作用や他のタグ付きタンパク質と第二、標識タンパク質（例えば、ポリオウイルス）との間の親和性に基づく信頼性の高い手法である。

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

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

a-liquid-phase-affinity-capture-assay-using-magnetic-beads-to-study

Research

JoVE Journal

Biology

11.8K ビュー.  Vrije Universiteit Brussel. この記事では、単純な、定量、液相の親和性捕捉アッセイが提示されています。それは一方では、磁気ビーズとタグ融合タンパク質（例えば、ナノボディ）との間の相互作用や他のタグ付きタンパク質と第二、標識タンパク質（例えば、ポリオウイルス）との間の親和性に基づく信頼性の高い手法である。

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

多重抗体スクリーニング法を用いたLuminex XMAPアッセイへの捕捉ELISAの変換

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

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 ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

中のタンパク質 - タンパク質相互作用を特定する<em&gt;ショウジョウバエ</emタンデムアフィニティー精製（TAP）による&gt;アダルトヘッド

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

ペプチド配列の使用タンパク質間相互作用部位を同定

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

IRE / IRP例：RNA  - タンパク質相互作用の研究のための電気泳動移動度シフトアッセイ（EMSA）

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

生細胞内のタンパク質断片相補アッセイ（PCA）によるゲノムワイドなタンパク質間相互作用のスクリーニング

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.