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><tbody><tr><td>Petri Dishes</td><td>VWR</td><td>NC-10747</td><td>or comparable</td></tr><tr><td>Bacto yeast extract</td><td>Difco</td><td>0217-17</td><td></td></tr><tr><td>Bacto peptone</td><td>Difco</td><td>0118-17</td><td></td></tr><tr><td>Bacto agar</td><td>Difco</td><td>0140-01</td><td></td></tr><tr><td>Dextrose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0514</td><td></td></tr><tr><td>Galactose</td><td>Sigma</td><td>G0750</td><td></td></tr><tr><td>Sc-Ura drop out media</td><td>MP Bio</td><td>114410622</td><td></td></tr><tr><td>Small Culture tubes</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Large Erlenmeyer Flask</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Centrifuge JA-10 rotor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (500 ml)</td><td></td><td></td><td></td></tr><tr><td>Falcon tube (50 ml)</td><td>VWR/Fisher</td><td>21008-951</td><td></td></tr><tr><td>PBS Tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>FastPrep Tubes (2 ml screw cap tube)</td><td></td><td></td><td></td></tr><tr><td>FastPrep Machine</td><td>MP Biomedicals</td><td>116004500</td><td></td></tr><tr><td>Zircon Beads</td><td>BioSpec</td><td>11079105</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014&amp;nbsp;</td><td></td></tr><tr><td>Imidazole (pH = 7.4)</td><td>Sigma</td><td>I5513&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>93482</td><td></td></tr><tr><td>Complete Inhibitor Cocktail (EDTA Free)</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail 1</td><td>sigma</td><td>P2850</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379&amp;nbsp;</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A1852&amp;nbsp;</td><td></td></tr><tr><td>Shaking Incubator (30 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Nickel Affinity Resin</td><td>Life Technologies</td><td>R901-01</td><td></td></tr><tr><td>G25 column</td><td>GE life sciences</td><td>27-5325-01&amp;nbsp;</td><td></td></tr><tr><td>Nutator</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>SDS-PAGE Gel (NuPage)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Coomassie Blue Stain</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Monoclonal V5 Antibody</td><td>Life Technologies</td><td>R960-25</td><td></td></tr><tr><td>Alexa647 Tagged monoclonal V5 Antibody</td><td>Life Technologies</td><td>451098</td><td></td></tr><tr><td>Protein Microarrays Kit</td><td>Life Technologies</td><td>PAH0525013</td><td></td></tr><tr><td>Genepix Scanner</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Lifter Slips</td><td>Thomas Scientific</td><td></td><td>http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td></tr><tr><td>Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT,&lt;br/&gt; 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&amp;nbsp; (add just prior to use)</td><td></td><td></td><td>Add the reagents to 1x 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><td>Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td><td></td><td></td><td>Add the reagents to 1x PBS to obtain the desired volume of blocking buffer</td></tr><tr><td>Probe Buffer: 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl, 500 &amp;mu;M ATP (for kinases), 1% BSA</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 &amp;ndash; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td><td></td><td></td><td>Before you begin, it is important to note the concentration gradient of imidazole (50 &amp;ndash; 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 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>3x YEP +6% galactose: Dissolve 30 g of yeast extract and&amp;nbsp; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td><td></td><td></td><td>&amp;nbsp;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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.0K 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

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低親和性受容体-リガンド相互作用のハイスループット検出の細胞外タンパク質マイクロ アレイ技術

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

生化学

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

ホスト病原体のタンパク質 - タンパク質相互作用の風景を特徴づけるために比較的アプローチ

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

免疫・感染学

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 ...

生物学

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

複雑なサンプル中の特定のタンパク質のグリコシル化の多重化ハイスループットプロファイリングのための化学的にブロックされた抗体マイクロアレイ

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

タンパク質相互作用を発見したHaloTag技術を用いたタンパク質機能の特性評価

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

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 ...

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 ...

蛍光を用いた化合物-タンパク質結合の高感度検出

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

著者スポットライト:アクセシビリティと効率性を向上させるためのPCR法の合理化(英語)

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

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

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低親和性受容体-リガンド相互作用のハイスループット検出の細胞外タンパク質マイクロアレイ技術

ホスト病原体のタンパク質 - タンパク質相互作用の風景を特徴づけるために比較的アプローチ

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

複雑なサンプル中の特定のタンパク質のグリコシル化の多重化ハイスループットプロファイリングのための化学的にブロックされた抗体マイクロアレイ

タンパク質相互作用を発見したHaloTag技術を用いたタンパク質機能の特性評価

ペプチド配列の使用タンパク質間相互作用部位を同定

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

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

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

要約

さらに動画を探す

この動画の章

低親和性受容体-リガンド相互作用のハイスループット検出の細胞外タンパク質マイクロ アレイ技術

ホスト病原体のタンパク質 - タンパク質相互作用の風景を特徴づけるために比較的アプローチ

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

複雑なサンプル中の特定のタンパク質のグリコシル化の多重化ハイスループットプロファイリングのための化学的にブロックされた抗体マイクロアレイ

タンパク質相互作用を発見したHaloTag技術を用いたタンパク質機能の特性評価

ペプチド配列の使用タンパク質間相互作用部位を同定

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

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

低親和性受容体-リガンド相互作用のハイスループット検出の細胞外タンパク質マイクロアレイ技術