This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1.プローブの準備<ol><li>培養し、以下のように他のタンパク質との相互作用を調べるために使用されるV5融合キナーゼプローブを精製します。 <ol><li>新しく作製した酵母株Y258を使用します（マタpep4-3、his4-580、ura3-53、leu2-3,112）（GATEWAYベクターpYES-DEST52から発現）V5融合タンパク質を含みます。マイクロアレイ上のプローブとして単離されたタンパク質を使用してください。合成完全ウラシル（SC-URA）/ 2％ブドウ糖/寒天に酵母をプレートと凍結培養（-80°Cグリセロールストック）から3日間30℃で成長します。 </li><li>単一コロニーからスターター培養物（5〜ml）を接種し、30℃でのプラットフォーム（220〜250 RPM）やホイールを振るのSC-URA / 2％デキストロース中で一晩成長します。 </li><li>翌朝は、0.1の最終OD 600に十分なスターター培養物を皮下-URA / 2％のラフィノース文化の400ミリリットルを接種します。 </li><li> 0.6の600は V5-キナーゼのfuのガラクトース誘導に続いてODまで接種を育てますシオンガラクトースの最終濃度が2％になるように3倍の誘導培地を希釈するのに十分に添加することにより、6％ガラクトースを補った3×酵母抽出物/ペプトン（YEP）の溶液を添加することによって表現を構築します。 </li><li>振盪プラットフォーム上で6時間30℃で細胞を誘導します。適切な通気を確保するために2 L三角フラスコを使用してください。 </li><li> 4℃で5分間、1000×gで細胞懸濁液を400mlの紡糸によってJA-10（または同等の）ローターを用いて収穫細胞。 </li><li>氷冷PBS緩衝液50mlで細胞を1回洗浄し、50mlコニカルチューブに移します。溶解のためのチューブキャップスナップ2ミリリットルに溶解し、転送に使用（洗剤または他の添加剤なし）の氷冷PBS緩衝液で再びペレットを洗浄。 </li><li>ペレットとピペット離れバッファに4℃で1分間20,000×gで細胞をスピン。 </li><li>氷の上にチューブを置き、溶解ステップに進みます。 </li><li> 0の細胞を溶解（250〜350μlのペレット）1：1 0.5ミリメートルのジルコニアビーズ1細胞ペレットの体積、ビーズ、およびリン酸緩衝生理食塩水（PBS）溶解緩衝液と4℃で2分間隔で3回攪拌プラットフォームを用いて混合物をボルテックス。 </li><li> 4℃で10分間、卓上微量20,000×gで溶解物を遠心します。 </li><li>ピペットポリカーボネート高速遠心チューブに上清を4℃で15万×gで30分間ulracentrufugationにより明確にします。 </li><li>ヒスチジンをキャプチャするために4℃で2時間、ニューテーター上で予備洗浄のNi 2+親和性樹脂（〜100μl）を含むチューブに清澄化ライセートを移し、インキュベート（彼の）6XがV5融合タンパク質をタグ付けしました。 </li><li>洗浄緩衝液で4℃で10分間樹脂を3回洗浄します。 4℃、1,000×gで5分間、卓上遠心機を用いて樹脂をペレット化し、上清を吸引します。各洗浄のために新鮮な洗浄緩衝液を添加し、洗浄の間にチューブを攪拌するための旋回装置を返します。 </li><li>洗浄後のSTEPSは、溶出のために新たにG-25カラムに洗浄した樹脂を適用し、完了しています。 </li><li> 9画分の合計のための50mM刻みで500 mMイミダゾールに100 mMのイミダゾールで始まる段階的に溶出バッファー25μlのを適用します。 </li><li>アッセイアッセイで使用するための最も高度に濃縮された画分を同定し、に使用するまで-80℃で30％ストアにグリセロールを添加することがドデシル硫酸ナトリウムポリアクリルアミドゲル（SDS-PAGE）およびクーマシー染色を用いて回収した画分アッセイ。 </li></ol></li></ol> 2.アレイのプロービング注：このアッセイを開始する前に、抗体溶液を調製するために、このセクションのステップ2.6を参照してください。 <ol><li>相互作用を検出するために、プローブ·バッファ内の260 ng / mlでのV5 -フルオロフォアコンジュゲート抗体（ すなわちのAlexaFluor 647）を希釈し、振とうして十分に混合します。 注：使用してnutatにチューブを配置する30分前に、この抗体溶液を準備しますやホイール完全混合し、抗体の均質な懸濁液を確保します。 </li><li> 5-500 / mlの濃度範囲にわたってV5融合タンパク質プローブを希釈します。 注：プローブ緩衝液を用いて、各タンパク質 - タンパク質相互作用アッセイのために最適化。最適化は、バッファをプローブするための複数のプローブを加えることを含みます。典型的には10μg/ mlの出発点として使用され、信号強度に基づいて、それに応じて調整されます。 </li><li>冷凍庫（-20℃）からタンパク質マイクロアレイを削除し、使用直前に冷蔵庫に4℃にもたらします。 </li><li>タンパク質micorarraysを含むスライドホルダーに直接ブロッキングバッファー追加し、漏出を防ぐためにパラフィルムでトップをカバーしています。 4℃でのステージ上で50 rpmで振とうすることによって1時間ブロッキング緩衝液中の配列をブロックします。 </li><li>ブロックした後、4℃に冷却加湿チャンバーに配列を転送し、アレイ表面に直接希釈したプローブの90μlを添加します。 ARRをオーバーレイ1.5時間4℃で加湿チャンバーで上昇リフタースリップでaysと静的インキュベート（無振盪）。 </li><li> 3 50mlコニカルチューブにプローブバッファに1分ごとにアレイを3回洗浄します。完全にスライドを包むのに十分な予備冷却プローブ緩衝液を含むコニカルチューブにスライドを追加します。リフタースリップを静かタンパク質マイクロアレイ（これはアレイ表面に損傷を与える可能性があるので、それを強制的にオフにしない）のオフにスライドすることができます。 </li><li>直前のような隆起したリフタースリップで洗浄（ステップ2.5）とオーバーレイを完了した後、アレイに直接抗体溶液を適用します。加湿チャンバー内で4℃で30分間、アレイをインキュベートします。 </li><li> （プローブ緩衝液中で3回1分）前と同じ洗浄工程を行い、室温で5分間、卓上遠心機で800×gで50mlのコニカルチューブをスピン。 647 nmでの配列をスキャンする前に30分間、暗所でスライドホルダーの配列を空気乾燥させます。 </li></ol>

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The authors declare that they have no competing financial interests.

プロトコルは提示本来インビボ 1 で新しいキナーゼ相互作用ネットワークの識別をもたらす酵母プロテインキナーゼの異なるファミリーおよび関連にわたって結合活性を比較するために85のユニークな酵母タンパク質キナーゼ-V5融合タンパク質を用いて行きました。新興プロテオミクスプロファイリング技術として、高スループット（HTP）ペプチドライブラリー、および...

タンパク質生化学およびインビボでの結合活性のグローバル分析を実行する必要性は、タンパク質-タンパク質相互作用（のPPI）と全プロテオーム1,3-8の翻訳後修飾をプロファイリングするための新たな方法の開発をもたらしました。タンパク質マイクロアレイは、全長機能タンパク質4-6,8,9、または抗体10,11を含む分析タンパク質マイクロアレイを用いた機能性タンパク質マイクロアレイとして製造されています。これらは、12を解析幅広い生化学的に行うために必要な実験条件の多様を容易にするために、表面化学の様々な顕微鏡スライド上に配列されたタンパク質の高濃度を含むように操作されています。リジンを介して化学結合またはそのような他の13の中親和性結合のためのHisタグ付きタンパク質およびグルタチオンを取り付けるためのニッケルキレート化スライドのような親和性結合方法のためのニトロセルロースとアルデヒド表面化学。 </p&gt; タンパク質-タンパク質相互作用を検出するための機能的なタンパク質マイクロアレイの使用は、高品質な機能性タンパク質のライブラリ14へのアクセスを必要とする。S.セレビシエは、ハイスループットクロマトグラフィー精製技術とタンパク質構築物をタグ付けし、高コピー親和性の対形成により、このようなライブラリーを作製に適しています。酵母ゲノムの大部分が、配列決定されており、ほぼ全プロテオームの精製および生化学12を分析するための高コピープラスミドから発現させることができます。タンパク質が得られ、384ウェル形式に配列されると、それらは迅速なパラレルマルチパラメトリック生化学分析およびバイオインフォマティクス尋問8,14-16を可能に顕微鏡スライド上にプリントされています。タンパク質マイクロアレイは、タンパク質、脂質、小分子、および他の多くの用途の中で核酸と酵素アッセイと相互作用するために使用されています。プロテオームの表面上のタンパク質のアクセシビリティアレイは、免疫​​親和性などの分析的検出の異なるタイプにそれらを敏感に反応する表面プラズモン共鳴、蛍光、および多くの他の技術を表面。また、それは、生体内で行うのは難しいかもしれない実験条件の微調整を可能にします。 このプロトコルの目的は、タンパク質 - タンパク質相互作用を検出するために、機能性タンパク質マイクロアレイの適切な使用を実証することです。このアプリケーションは、目的の高度に精製された分析物（タンパク質）を用いてタンパク質結合活性のハイスループット並列生化学的解析を可能にします。 C末端（カルボキシ末端）は、関心のV5融合ベイトタンパク質は、タンパク質精製のために最適化された酵母株における高コピープラスミドから生成されるタグ付き。 C末端タグは、全長タンパク質が翻訳されていることを保証します。この研究で使用されるタンパク質は、His6Xタグを介して、ニッケル親和性樹脂を用いて精製するTda1-V5融合タンパク質キナーゼです。 Tda1-V5融合constructは、アッセイで使用するための最も高度に濃縮された画分を溶出するためにイミダゾール勾配を使用して、シリアル溶出により精製されます。

<table border="0" cellpadding="0" cellspacing="0" width="855">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Petri Dishes</td>
			<td style="width:180px;">VWR</td>
			<td>NC-10747</td>
			<td>or comparable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto yeast extract</td>
			<td style="width:180px;">Difco</td>
			<td>0217-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto peptone</td>
			<td style="width:180px;">Difco</td>
			<td>0118-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:180px;">Difco</td>
			<td>0140-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dextrose</td>
			<td style="width:180px;">Sigma</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Raffinose</td>
			<td style="width:180px;">Sigma</td>
			<td>R0514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Galactose</td>
			<td style="width:180px;">Sigma</td>
			<td>G0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sc-Ura drop out media</td>
			<td style="width:180px;">MP Bio</td>
			<td>114410622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Small Culture tubes</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Large Erlenmeyer Flask</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge JA-10 rotor</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge tubes (500 mL)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Falcon tube (50 mL)</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PBS Tablets</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Tubes(2ml screw cap tube)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Machine</td>
			<td style="width:180px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">116004500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Zircon Beads</td>
			<td style="width:180px;">BioSpec</td>
			<td align="right" style="width:119px;">11079105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Triton X100</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DTT</td>
			<td style="width:180px;">Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">MgCl2</td>
			<td style="width:180px;">Sigma</td>
			<td>M8266&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">NaCl</td>
			<td style="width:180px;">Sigma</td>
			<td>S3014&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Imidazole (pH = 7.4)</td>
			<td style="width:180px;">Sigma</td>
			<td>I5513&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PMSF</td>
			<td style="width:180px;">Sigma</td>
			<td>93482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Complete Inhibitor Cocktail (EDTA Free)</td>
			<td style="width:180px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Phosphatase inhibitor cocktail 1</td>
			<td style="width:180px;">sigma</td>
			<td>P2850</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSA</td>
			<td style="width:180px;">Sigma</td>
			<td>A2153&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Tween-20</td>
			<td style="width:180px;">Sigma</td>
			<td>P1379&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ATP</td>
			<td style="width:180px;">Sigma</td>
			<td>A1852&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Shaking Incubator (30 &#176;C)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nickel Affinity Resin</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R901-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">G25 column</td>
			<td style="width:180px;">GE life sciences</td>
			<td>27-5325-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nutator</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">SDS-PAGE Gel (NuPage)</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Coomassie Blue Stain</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R960-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa647 Tagged monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>451098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Protein Microarrays Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td>PAH0525013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Genepix Scanner</td>
			<td style="width:180px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:267px;">Lifter Slips</td>
			<td style="width:180px;">Thomas Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:291px;">http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td>
		</tr>
		<tr height="124">
			<td height="124" style="height:124px;width:267px;">Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT, 
			2 mM MgCl2, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&#160; (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS to obtain the desired volume of blocking buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Probe Buffer: 2 mM MgCl2, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl,500 &#956;M ATP (for kinases), 1% BSA</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:267px;">Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl2</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:267px;">Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl2, 500 mM NaCl, 50 &#8211; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Before you begin, it is important to note the concentration gradient of imidazole (50 &#8211; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:267px;">3x YEP +6% galactose: Dissolve 30 g of yeast extract and&#160; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;galactose should not be autoclaved</td>
		</tr>
	</tbody>
</table>

probing high-density functional protein microarrays to detect protein-protein interactions

〜4,200組換え酵母タンパク質を含む高密度機能タンパク質マイクロアレイは、読み出しのためのV5エピトープタグを含むアフィニティー精製酵母キナーゼ融合タンパク質を用いてキナーゼタンパク質 - タンパク質相互作用のために検査されます。精製されたキナーゼは、GAL、誘導性プロモーター下キナーゼ-V5融合構築物を含むプラスミドを有する高コピータンパク質産生のために最適化された酵母株の培養により得られます。酵母は2％ガラクトースで誘導し、続いて6時間、中性の炭素源と制限された培地中で成長させます。次に、培養物を回収し、キナーゼアッセイにおける使用のための高度に精製されたタンパク質キナーゼを得るために、標準的なアフィニティークロマトグラフィー技術を用いて精製します。精製されたキナーゼアッセイのために適切な範囲のキナーゼ緩衝液で希釈し、タンパク質マイクロアレイは、タンパク質マイクロアレイにハイブリダイゼーションの前にブロックされます。ハイブリダイゼーション後、アレイをモノクローナルV5 antibでプローブしますキナーゼV5タンパク質によって結合したタンパク質を同定するODY。最後に、アレイは、標準的なマイクロアレイスキャナーを用いてスキャンされ、データは、インビボでの下流の検証のためのタンパク質の相互作用の高い信頼度のセットを決定するために、下流インフォマティクス分析1,2のために抽出されます。

タンパク質-タンパク質相互作用活性は、約4,200ユニークS.を含む酵母機能的タンパク質マイクロアレイに対するベイトタンパク質として構築Tda1-V5タンパク質キナーゼ融合を評価するために、標準的なチップリーダーを用いて観察されましたセレビシエ GST融合タンパク質。 GENEPIXソフトウェアとさらに尋問は、様々な強度の結合事象の多くを明らかにしました。親和性は、その標?...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

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

ほぼ全Sを含むタンパク質マイクロアレイを用いて、 セレビシエプロテオームは、並行して、タンパク質-タンパク質相互作用の何千もの迅速公正な尋問のためにプローブされます。この方法は、タンパク質 - 小分子、翻訳後修飾、およびハイスループットの他のアッセイに利用することができます。

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 ...

probing-high-density-functional-protein-microarrays-to-detect-protein

Research

JoVE Journal

Biology

7.9K ビュー.  Stanford University. ほぼ全Sを含むタンパク質マイクロアレイを用いて、 セレビシエプロテオームは、並行して、タンパク質-タンパク質相互作用の何千もの迅速公正な尋問のためにプローブされます。この方法は、タンパク質 - 小分子、翻訳後修飾、およびハイスループットの他のアッセイに利用することができます。 

ビデオ: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

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

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 ...

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, ...

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 ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

化学的に媒介種間相互作用を検出するための共培養を使用して

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

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 ...

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 ...

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 ...