私たちは、私たちに酵母のTAP発現プラスミドを送信するためEUROSARFに感謝します。また、ライアンLabadensから編集助けに感謝しています。この作品は、CWにNIH / NINDS助成金（R01NS070962）によってサポートされていました

1。 UAS-TAPタグトランスジェニックハエを生成<ol><li>のpUAST-TAPタグDNA構築物を生成します。 <ol><li> TAPタグはタンパク質の構造/機能に基づいて、に融合されるべきベイトタンパク質のどちら側（N-またはC-末端）を決める。詳細についての説明を参照してください。 </li><li>それぞれ、N-またはC-末端タグ付加UAS-TAPトランスジーンを生成するためのpUAST-又はNTAPのpUAST-CTAPベクターのマルチクローニング部位（MCS）への目的の遺伝子のcDNAコード領域をサブクローニングする。詳細な地図と使用可能な制限部位とリーディングフレームについては、図1を参照してください。 </li></ol></li><li> UAS-TAPタグトランスジェニックハエを生成します。 <ol><li> Pエレメント媒介挿入11を使用して、標準的なプロトコルに従ってトランスジェニックハエを生成します。注射サービスの数が市販されている。 </li><li>それぞれの個々のトランスジェニック系統に神経GAL4ドライバー（ すなわち、BG380-GAL4）を渡り、タンパク質の発現レベルを決定する（ペルオキシダーゼ抗ペルオキシダーゼ抗体を用いた）ウエスタンブロットによる各ラインのおよび/または（抗TAP抗体で）免疫染色。一般的には、内因性タンパク質に近いタンパク質発現レベルを有するトランスジェニックラインは、TAPの手続きをお勧めします。詳細についての説明を参照してください。 </li><li>目的の遺伝子の機能喪失変異体が利用可能な場合は、TAPタグ化導入遺伝子の機能性を確認するためのレスキュー実験をPerformGAL4/UAS-based。ほぼ次のTAP実験に変異体の表現型を救出することができ、導入遺伝子を選択してください。 </li></ol></li></ol> 2。 TAP手順のためのサンプルを調製<ol><li>フライサンプルの拡大を容易にするために、神経Gal4ドライバー（ 例：BG380-GAL4）と選択したTAPタグ導入遺伝子の両方を運ぶハエの株式を生成します。 GAL4ドライバと上記の組み合わせは生存と成長の欠点の原因となるまれにUAS-導入遺伝子のクロスのF1子孫を収集します。&lt;/李&gt; <li>小規模のサンプルを収集して、TAPタグ化タンパク質を可溶化するための溶解条件を最適化します。 <ol><li>非イオン性界面活性剤NP-40（0.1％）、NaDOC（0.1％）およびTriton X-100（0.05から0.5パーセント）の組み合わせを用いて溶解緩衝液のシリーズを作る。詳細については、表1および説明を参照してください。 </li><li> CO2のパッドの上に、ハエを表現TAPトランスジーンから20大人の頭を解剖し、各溶解バッファー条件をテストするために1.5ミリリットルチューブでそれらを収集するために＃5鉗子を使用しています。 </li><li>チューブに100μlの試験溶解バッファーを追加し、プラ​​スチック乳棒で上下になでるとで頭を均質化し、他の100のULテストバッファを追加します。 </li><li> 10分（4℃）のために21,500×gで頭溶解物を回転し、遠心分離後の上清とペレットを分離する。それぞれの上清の30 ULにペレットを10μlの4X SDSローディングバッファに25 ulの2X SDSローディングバッファーを追加します。 </li><li> 5分間の2サンプルを沸騰して並べ、それらを分析側PAP抗体でSDS-PAGEおよびその後のウエスタンブロットを用いて。ペレットVS上清中のTAP-タンパク質レベルの比で溶解度を決定する。 </li></ol></li><li>大規模なサンプルを作成<ol><li>大人のハエを展開し、収集します。 <ol><li>ボトルにneuronal-GAL4/UAS-TAP-transgeneの株式を展開し、累積250ボトル収集に使用されるまで、3日ごとにボトルを反転させます。 </li><li>収集1-3日齢の成人が50ミリリットルコニカルチューブに飛んで、すぐにDeep Freezeのハエに液体窒素中にチューブを入れる。 -80℃の冷凍庫にハエを格納します。ハエの量は50ミリリットルチューブの2/3を超えてはならないことに注意してください。 </li></ol></li><li> （粉末ドライアイスの上に、この手順を実行する）フライヘッドを収集します。 <ol><li>予冷キュラーシーブおよび-80℃の冷凍庫から、乳鉢と乳棒を取り出して、理想的には大規模な氷のバケツの中に、ドライアイス上に置く。日に第25号2米国標準試験ふるいを積み重ねEの上下に40番。 </li><li>凍結したハエを取り出し、液体窒素にドロップし、約10分間そこにハエを保つ。ボルテックスまたは体から頭、脚、翼を破るために積極的にチューブを横に振る。 </li><li>トップふるいに混合を注ぎ、その後、一緒に両方のモレキュラーシーブを押しながら積極的にモレキュラーシーブを振る。篩い分けした後、遺体は、トップふるいに滞在ヘッドが下ふるい上に保持されます飛ぶ、と脚、翼、および他の残骸は、ドライアイスに落ちるでしょう。 2モレキュラーシーブを分離し、慎重に冷たい乳鉢にハエの頭を転送します。 </li></ol></li><li>フライヘッドを均質化する<ol><li>ドライアイスの上に、氷の上にあらかじめ冷却した15ミリリットルのガラスダウンス組織グラインダーに粉を転送した後、粉末状の粒子に乳鉢と乳棒で頭を挽く。 </li><li>ヘッドサンプルがそれに注いだの前後グラインダーの重量を測定してから、どのくらいのヘッドSAMP計算LEは重さがあります。ハエ頭部6-15グラムの総タンパク質の発現レベルに応じて調整する各TAP実験のために十分であろう。乳棒を上下に行くとすることが容易であるまで、粉末に氷冷した均質化緩衝液の15ミリリットル（ステップ2.2に最適化された溶解バッファー）を追加してから、大きなクリアランス乳棒でストローク。すべての回で、氷上でガラス粉砕機を保管してください。 </li></ol></li><li> TAPのために上清を準備<ol><li> 〜5万×gで（4℃）で20分間の高速遠心分離管とスピンにホモジネートを転送する。新しい高速遠心チューブに上清を移し、遠心分離をもう一度繰り返します。 </li><li>超遠心チューブに上清を移し、さらに上清をクリアするには40分〜25万XGスピンを行う。上清を超遠心分離後のタンデムアフィニティー精製法の準備ができています。 </li></ol></li></ol></li></ol> 3。 TAP精製</p&gt; 以下のセクションでは、セラフィンラボタップから導出されたprotocol 12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf ） <ol><li>ビーズのIgGアフィニティー精製を行う<ol><li>サンプルは遠心分離されている間のIgGセファロースビーズを準備します。 10ミリリットル冷たいのIgG洗浄緩衝液15ミリリットルファルコンチューブに400μLのIgGビーズ3回洗浄します。各洗浄のために、2分間穏やかチューブを揺するし、別の2分間千×gでビーズをスピンダウン。 3回目の洗浄の最後に、緩衝液を除去し、チューブ内のビーズのみを残す。 </li><li>注意深くのIgGビーズを含有する15mlチューブに透明な上清（〜15ml）に移す。 2時間旋回装置上で4℃でビーズや脳溶解ミックスをインキュベートする。 </li><li>低温室で約15ミリリットルの総容量で、清潔で、空のミクロカラムを設定します。着実に塔にミックスを注ぐことにより、のIgGビーズ混合物をロードし、あらゆるトラップしないようにしよう空気が列の中に泡。ビーズはゆっくりと重力流によって排出する列バッファに定住することができます。 </li><li>脳溶解物のすべてが決着IgGカラムを流れた後に冷えたIgGの洗浄緩衝液10mlで十分にカラムを洗浄。洗浄2Xを繰り返します。注意：空気中のビーズ乾燥させることはありません。 </li></ol></li><li> TEV切断を行う<ol><li> 3回目の洗浄後、10ミリリットルTEV切断緩衝液で再びカラムを洗浄。このステップは、TEV切断用の餌複合体をsequestratesのIgGビーズを準備します。 </li><li> TEVバッファの最後の一滴が、出ドリップ流れを遮断する列の一番下にキャップを入れて、列にTEV酵素の130単位を含有する1.3ミリリットルTEVバッファーを追加して、安全に上部のキャップをしようとしている直前にコラム。列は両端でよく密封されていることを確認してください。 </li><li> TEV酵素はTEV部位でペプチドを切断およびタンパク質複合体WHIのを放出できるようにするために2時間18度Cでカラムを回転させるルタンパク質の背後のIgGセファロースビーズに結合ドメインペプチドを残す。 </li></ol></li><li>カルモジュリンビーズアフィニティー精製を行う<ol><li> IgGのビーズをTEV酵素とインキュベートしている間にカルモジュリンビーズを準備します。冷たいカルモジュリン結合バッファー10mlで毎回、15ミリリットルファルコンチューブ3Xに200μlのカルモジュリンビーズを洗浄します。各洗浄のために、ゆっくりと旋回装置上で2分間、チューブを揺らし、その後、2分間千×gでビーズをスピンダウン。 3回目の洗浄の最後に、すべてのバッファーを取り出して試験管内のビーズのみを残す。 </li><li> TEVのインキュベーション（ステップ3.2.3）の終了時に、戻って寒い部屋にIgGカラムを返し、まっすぐ上に設定します。ビーズは、10分のために解決しましょう​​。 </li><li>トップキャップを外し、ボトムキャップした後、15ミリリットルファルコンチューブ内の1.3ミリリットルのTEV切断産物を収集します。バッファが完全に排水してみましょう。列のデッドボリュームを押し出す列に追加の200μlのTEVバッファーを追加し、Fを収集同一チューブ内の低アウト。 </li><li>上記の収集された1.5ミリリットルTEV切断製品にバッファし、4.5μlの1 M 塩化カルシウムカルモジュリン結合の4.5ミリリットルを追加します。 塩化カルシウムは、TEVバッファー中のEDTAを滴定するのに役立つ。カルモジュリンビーズを含むチューブに6ミリリットルの混合物を移し、1時間旋回装置上で4℃でチューブを回転させます。 </li><li>低温室で約10ミリリットルの総容量を持つ別の清潔で、空のミクロカラムを設定します。列にカルモジュリンビーズ混合物をロードし、それが重力によって排出させる。 </li><li>すべてのソリューションが確定カルモジュリンカラムに流れた場合には、列に冷たいカルモジュリン結合緩衝液10mlでそれぞれ2回洗浄する。注意：カルモジュリンビーズを乱さないよう、洗浄の際に、可能な限りフラットなビーズの表面を維持しようとします。 </li></ol></li><li>カルモジュリン列から餌複合体を溶出させる。 右洗濯後、200μlの冷たいCalmoduの5画分とカルモジュリンカラムを溶出LINの溶出バッファー。各分画のために、ゆっくりとカラムに溶出緩衝液200μlを追加し、マークされた1.5mlのエッペンドルフチュー​​ブに溶出液を収集します。この4倍を繰り返します。 </li><li> SDS-PAGEによるタンパク質複合体の分析<ol><li> 5分（約30μL）のそれぞれから少量をとり、SDSローディングバッファーを追加します。 5分間のサンプルを沸騰させ、勾配（4から15パーセント）のSDS-PAGEゲル中のタンパク質の分子マーカーを有するサンプルを並べてロードします。 </li><li>サンプルは完全にゲルで解決した後、電気泳動を停止し、そのような13の染色「ブルーシルバー」などの任意のG-250をベースに敏感コロイド状クマシー染色手順でゲルを染色。それは、その後の質量分析と完全に互換性がないため、銀染色はオプションですが、好ましくない。このような精製されたタンパク質複合体の分子のアイデンティティを発見するための質量分析などのさらなる分析のために-80℃の冷凍庫に溶出液の残りの部分を格納します。 SEEディスカッション。 </li></ol></li></ol>

<ol>
	<li>Rigaut, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat. Biotechnol. 17, 1030-1032 (1999).</li><li>Collins, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+comprehensive+atlas+of+the+physical+interactome+of+Saccharomyces+cerevisiae.">Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.</a> Mol. Cell. Proteomics. 6, 439-450 (1074).</li><li>Gavin, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+survey+reveals+modularity+of+the+yeast+cell+machinery.">Proteome survey reveals modularity of the yeast cell machinery.</a> Nature. 440, 631-636 (2006).</li><li>Krogan, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+landscape+of+protein+complexes+in+the+yeast+Saccharomyces+cerevisiae.">Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.</a> Nature. 440, 637-643 (2006).</li><li>Forler, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+protein+complex+purification+method+for+functional+proteomics+in+higher+eukaryotes.">An efficient protein complex purification method for functional proteomics in higher eukaryotes.</a> Nat. Biotechnol. 21, 89-92 (2003).</li><li>Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+complexes+in+Drosophila+with+tandem+affinity+purification-mass+spectrometry.">Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.</a> Dev. Dyn. 232, 827-834 (2005).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+technology:+an+overview.">The tandem affinity purification technology: an overview.</a> Biotechnol. Lett. 33, 1487-1499 (2011).</li><li>Volkel, P., Le Faou, P., Angrand, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+proteomics:+characterization+of+protein+complexes+using+tandem+affinity+purification-mass+spectrometry.">Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry.</a> Biochem. Soc. Trans. 38, 883-887 (2010).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+method:+an+efficient+system+for+protein+complex+purification+and+protein+interaction+identification.">The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.</a> Protein Expr. Purif. 72, 149-156 (2010).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Bachmann, A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+P-element+transposons+to+generate+transgenic+flies.">The use of P-element transposons to generate transgenic flies.</a> Methods Mol. Biol. 420, 61-77 (2008).</li><li>Puig, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 (2001).</li><li>Candiano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+silver:+a+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25, 1327-1333 (2004).</li><li>Tian, X., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ubiquitin-mediated+pathways+in+regulating+synaptic+development,+axonal+degeneration+and+regeneration:+insights+from+fly+and+worm.">The role of ubiquitin-mediated pathways in regulating synaptic development, axonal degeneration and regeneration: insights from fly and worm.</a> J. Physiol. , (2013).</li><li>Wu, C., Wairkar, Y. P., Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highwire+function+at+the+Drosophila+neuromuscular+junction:+spatial,+structural,+and+temporal+requirements.">Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.</a> J. Neurosci. 25, 9557-9566 (2005).</li><li>Wu, C., Daniels, R. W., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DFsn+collaborates+with+Highwire+to+down-regulate+the+Wallenda/DLK+kinase+and+restrain+synaptic+terminal+growth.">DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth.</a> Neural Dev. 2, 16 (2007).</li><li>Tian, X., Li, J., Valakh, V., DiAntonio, A., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Rae1+controls+the+abundance+of+the+ubiquitin+ligase+Highwire+in+post-mitotic+neurons.">Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons.</a> Nat. Neurosci. 14, 1267-1275 (2011).</li><li>Burckstummer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+tandem+affinity+purification+procedure+for+interaction+proteomics+in+mammalian+cells.">An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.</a> Nat. Methods. 3, 1013-1019 (2006).</li><li>Kyriakakis, P., Tipping, M., Abed, L., Veraksa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+in+Drosophila:+the+advantages+of+the+GS-TAP+system.">Tandem affinity purification in Drosophila: the advantages of the GS-TAP system.</a> Fly. 2, 229-235 (2008).</li><li>Bailey, D., Urena, L., Thorne, L., Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+interacting+partners+using+tandem+affinity+purification.">Identification of protein interacting partners using tandem affinity purification.</a> J. Vis. Exp. (60), e3643 (2012).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+for+Angelman+syndrome.">A Drosophila model for Angelman syndrome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 12399-12404 (2008).</li><li>Liao, L., McClatchy, D. B., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+proteomics+in+neuroscience.">Shotgun proteomics in neuroscience.</a> Neuron. 63, 12-26 (2009).</li></ol>

著者らは、開示することは何もありません。

タンデムアフィニティー精製（TAP）法2の独立したアフィニティー精製工程を経てタンパク質複合体の単離及び濃縮を可能にするデュアル精製プロトコルを提供しています。 TAPタグの設計は、このプロトコルで提示されるものに限定されない緩衝液条件に応じて調整される場合、他のタンパク質の結合ドメインおよびモチーフも適用可能である。他のTAPタグの良い例は、哺乳類の培養細胞株<s...

特定の生物学的プロセスを仲介する分子経路やネットワークを定義することは、生物医学研究の究極の目標の一つである。フライ遺伝学者は、特にと並行して、一緒に作業したり、上流または関心のある遺伝子の下流因子を同定するために、遺伝子スクリーニング（エンハンサーとサプレッサー画面の両方を）修飾子、フォワード遺伝学に大きく依存してきた。しかし、フォワード遺伝学の画面はしばしば、その損失関数の唯一の得点にくい微妙な異常を引き起こす機能的冗長性と補償で不可欠な変異した場合に、初期の発達段階で致死性の原因となる遺伝子、または遺伝子を同定することができない。この困難を克服する1つの方法は、直接的なタンパク質 - タンパク質相互作用をスクリーニングするためのものである。 10年以上のため、 などの酵母ツーハイブリッド、ファージディスプレイ、化学的架橋、共同IP、タンデムアフィニティー精製（TAP）を含む生化学的方法、の成長のリスト。タンパク質を調査するために使用されている- タンパク質相互作用。これらのアプローチはそれぞれ長所と感度と特異性に関しての弱点の独自のセットを持っています。中でも、TAP法は、近生理学的条件下で物理的相互作用の検出を可能にする特異性および整合性を維持し、2 3,4を解析し、高スループットを拡張する能力を含む。 TAP法は、もともとRigautand同僚1によって、酵母で開発されました。この方法では、対象のタンパク質は、TAPタグで表現される。 TAPタグは、2つの独立した親和性結合ドメインを保有する：タンパク質IgGおよびカルモジュリン結合ドメインに結合するドメイン。 2つのドメインはTEV（タバコエッチウイルス）切断部位によって分離されている。アフィニティ精製の ​​二つの独立したラウンドが十分に非特異的結合を減少させ、特定のバインディング1を豊かにするためのこのような組み合わせが可能です。この例では、TAP法は非常に強力であるメト外因性タンパク質を過剰発現すると、通常、その内因性の相手との複雑なないタンパク質と結合することをより受けやすいことがありますが、与えられたタンパク質の生体内相互作用に識別するためのD。その開発以来、TAP法は、in vivoモデル系6-9 の細胞培養ベースのシステム5,6およびその他を含む多くの他のシステムに適用されている。ここでは、 ショウジョウバエのTAP法の適応を説明します。まず、目的の遺伝子のN末端またはC末端のいずれかに、TAPタグのクローニングとの融合を容易にするためのpUAST-NTAPとのpUAST-CTAPベクトルを生成する。 UAS-TAP-タグ化導入遺伝子は、次いで、ニューロンGAL4ドライバ10の制御下で、神経系で発現される。次に、成体ハエの頭部に多数の神経細胞の高い含有量を有​​し、サイズの違いに基づいて、凍結後に体の他の部分から分離することが容易である、収集することができる。大人のヘッドは、均質化され、BをクリアYシーケンシャル遠心分離し、上澄み液は、後述のTAPの手順に従うものとします。

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">U.S.A. standard test sieve No. 25</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-18&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Kontes Dounce Tissue Grinders 15 ml</td>
			<td>Kimble Chase</td>
			<td>885300-0015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;"></td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG sepharose beads</td>
			<td>Pharmacia</td>
			<td style="width:119px;">17-0969-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.7 cm x 20 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4721</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.5 cm x 15 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4716</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calmodulin beads</td>
			<td style="width:104px;">Stratagene</td>
			<td style="width:119px;">214303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coors Mortar and Pestle</td>
			<td style="width:104px;">CoorsTek</td>
			<td>60311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AcTEV Protease</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:104px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Mix</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

キイロショウジョウバエ （ショウジョウバエ）を用いて行っ遺伝子スクリーニングは、生物科学の前に数々のマイルストーンの発見をした。しかし、遺伝学的解析から得られた知識を広げることを目的とし、生化学的スクリーンの使用は、ごく最近になって調査した。ここでは、成体ハエ頭部から目的の任意のタンパク質と会合したタンパク質複合体を精製する方法を記載している。この方法は、 インビボでのフライニューロンにおけるタンデムアフィニティー精製（TAP）タグと融合ベイトタンパク質を発現するために、ショウジョウバエ GAL4/UASシステムを活用し、その後、本来において確立ものと類似のTAP手順を用いた精製を2回を実装相互作用するタンパク質複合体を精製する酵母1。この手順の最後に、複数のタンパク質複合体の混合物は、その分子のアイデンティティ質量分析によって決定することができるが得られる。候補タンパク質の検証は、Rの恩恵を受けるesourceやハエにおける機能喪失研究を行うのしやすさ。同様のアプローチは他のハエ組織に適用することができる。我々はフライモデル系における遺伝子操作の組み合わせと、このプロテオミクスアプローチは、神経生物学とそれ以降の分野で根本的な問題に取り組むための途方もない可能性を秘めていると信じています。

ここでは、ハエの脳内のHighwire相互作用タンパク質を同定することに我々の努力を示しています。 Highwire（HIW）とその脊椎動物と無脊椎動物の同族体は、神経系14の開発と修復を規制する巨大なユビキチンリガーゼである。彼らは高度に保存された機能的なドメインの数を共有しています。しかし、それらの分子の行動は完全には明らかではありません。ワームで行われた作業、飛ぶ?...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP at JoVE.com

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

ショウジョウバエは、その強力な遺伝子操作のためではなく、詳細な生化学的解析のその適合性のために有名である。ここでは、ハエの脳から、目的の任意のタンパク質の相互作用パートナーを識別するために、TAPベースの手順を提示する。この手順では、潜在的な研究の新たな道につながることができます。

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-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

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

13.8K ビュー.  Louisiana State University Health Sciences Center.  ショウジョウバエは、その強力な遺伝子操作のためではなく、詳細な生化学的解析のその適合性のために有名である。ここでは、ハエの脳から、目的の任意のタンパク質の相互作用パートナーを識別するために、TAPベースの手順を提示する。この手順では、潜在的な研究の新たな道につながることができます。 

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

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

小型二重特異性アフィニティタグによって容易に直交するタンパク質の精製

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

生物学

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

タンデムアフィニティー精製を用いたタンパク質相互作用するパートナーの識別

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

転写産物および代謝物からの精製<em&gt;ショウジョウバエ</em&gt;ヘッド

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

マイクロスケール熱泳動によって結合親和性の決定のタンパク質精製·フリー法

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...