この作品は、健康補助金GM086822と国立科学財団の主要な研究助成金計装DBI-0960313の国立研究所によって資金を供給された。著者は、博士に感謝します。ジョン·三宅＆ドナハーディ（Bio-Rad社）とその技術的専門知識のためにジェニファーLoertscher（シアトル大学）。クロマトグラフィー用試薬を選択し、補足的な計装は、寛大にBio-Rad社によって提供されました。

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クロマトグラフィー用試薬を選択し、補足的な計装は、Bio-Radから提供されました。

液体クロマトグラフィー技術は、7生化学的免疫学6を行うために必要な高度に精製したタンパク質、および構造的な8つの研究を準備するために貴重な実証されています。 HICの精製方法は、ほとんどの場合、好ましい培地の経験的判断を必要とし、配位子の構造、リガンド密度、マトリックスビーズのプロパティはすべてのクロマトグラフの結果は2、3に影響を与えることが示されている。スカウト自動化された列は、その後の最適化とタンパク質精製4 HIC培地を選択するための効率的なアプローチである。提示自動化されたカラムスカウティング手法は、容易に様々なHICタンパク質の精製戦略に適合させることができます。塩濃度、塩の選択は、塩勾配、およびpHの変化は、さらに精製条件を向上させることができ、これらの必須のパラメータを変えることの効果は以前に13,14日されています。部分的に精製されたターゲットproteを含むHIC溶出液には、さらに、イオン交換クロマトグラフィー（IEX）またはゲル濾過/サイズ排除クロマトグラフィー9,10などの補完的なクロマトグラフィー技術を用いて精製することができます。 そのユニークな光の吸光度および発光特性の、GFPの溶出プロファイル内の行と、実行後のアプローチを使用して識別することができます。そのために、このプロトコルは、さらに関係のない目的タンパク質がGFPで &quot;タグ付け&quot;されている、組換えGFP融合タンパク質の精製に適応することができます。 GFPタグ付きの標的タンパク質は、UV光11と、上記HIC精製戦略を含む様々なクロマトグラフィー手法を用いて精製して検出することができます。 GFPの精製はまた、近代的な蛋白質科学技術の12の教育のための生化学ラボで教育主食となっています。 基本的なHICタンパク質の精製は、実質的に少ない堅牢なクロマトグラフィーSYを用いて行うことができますが、ステムは、ここで紹介するインストルメンテーションは、良好な結果を得ることを支援するために、異なる多くの利点があります。高度に自動化システムを利用しての顕著な利点は、改善·実行·実行するために条件の再現性、時間を節約し、システム10に導入される空気の機会を減少させたが含まれています。システム·マキシマイザーにより促進されるシステム制御バッファブレンディングは、バッファの準備と実験の再現性に増加した一貫性を可能にします。スカウトは、さらに実行するすべての動的なサンプルループに十分なサンプルの負荷を描画すると、サンプルの均一性は、各列に追加され確保され、中断、または手動でリロードすることなく、順次実行を可能にします。カラムにサンプルループから制御された試料注入は、手動でサンプルロードで発生する可能性がありばらつきが減少します。カラム選択バルブのペアは、それぞれのシステムをreplumbすることなく、異なるHICカラムを使用して、連続したサンプルの実行が可能になります。 MULを実行する<html>ユニークな分光プロファイルを使用して蛋白質を測定する時TI-波長分析では、特に有益です。 GFPに加えて、シトクロム、flavoproteins、および他のヘム含有タンパク質は、この技術の恩恵を受ける可能性があります。伝導度とpHモニタリングデバイスは、リアルタイムの実験条件の検証を可能にします。分割分数溶出コレクションは改善された分画処理を行うことができ、最小限のサンプル量（例えば、ELISA、SDS-PAGE、ウェスタンブロット、とExperionマイクロ流体電気泳動）を必要と実行後の分析手法について簡単に転送できます。第二のフラクションコレクタをオフラインで操作すると手動で同期を必要とするが、それはフラクションコレクタの選択に最大の柔軟性を可能にします。 HICスカウトカラムとタンパク質の精製のためにそのような堅牢なクロマトグラフィーシステムを活用した最も重要な欠点は、楽器の収集とオペレータのトレーニングに関連付けられている最初の時間と予算の支出が含まれています。 T &quot;&gt;ここで紹介するプロトコルは、Bio-Rad社DuoFlowクロマトグラフィーシステムを利用します。。しかし、このようなGEヘルスケアからÄKTAの前衛のような他のメーカーからも同様に堅牢なインストルメンテーションを利用することもできると同等の結果を生成することが可能であっても同等のクロマトグラフィーシステム精製法や楽器の取得を開始する前に考慮すべき固有の特性（例えば、メソッドのプログラミング、コンポーネントの命名法、演算子の優先順位、およびスケーラビリティの制限）があります。

<table border="1"><tbody><tr><td>試薬の名前</td><td>会社</td><td>カタログ番号</td><td>コメント（オプション） </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">生物学的DuoFlowパスファインダー20システム</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">7602257</a> </td><td>システムマキシマイザー、ミキサー、F10のワークステーション、AVR7-3バルブ、フローセル、BioFracフラクションコレクター、生物学的ソフトウェア、スターターキットを使用QuadTecのUV / VIS検出器を含む</td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">AVR9-8ストリーム選択バルブ</a><html></td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7600408</a> </td><td>生物学的DuoFlowシステムで使用する高圧バルブ、9ポート、8ポジション、3,500 PSI（233バー）の制限、 </td></tr><tr><td>分流弁SV3T-2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> 7600410 </td><td>生物学的DuoFlowシステムで使用する電磁弁、3ポート、2ポジション、30 PSI（2バー）の制限、 </td></tr><tr><td>ストリームスプリッタバルブ</td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td></td><td></td></tr><tr><td>tical = LSR＆parentCategoryGUID = 22c495f1-36ff-44a3-9140-ee7e54d0b1ba &quot;ターゲット=&quot; _blank &quot;&gt; DynaLoop 25キット</td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7500451</a> </td><td>スライディングピストンサンプルループ·キット、25ミリリットルのDynaLoopスライディングピストンループ、DynaLoop部品キット（＃750から0450まで）が含まれています</td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">BioFracフラクションコレクタ</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">7410002</a> </td><td>二つの分数COL<html>使用lectors </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">BioFracマイクロドロップヘッドアダプタ</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">7410088</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">BioFracマイクロタイタープレートアダプタ</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td>＆国=米国＆LANG = EN＆ProductID =に741-0017 &quot;ターゲット=&quot; _blank &quot;&gt; 7410017 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">UV光モジュール</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">7500202</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">ハロゲンランプ</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> &lt;<html> 1ターゲット= &quot;_blank&quot;&gt; 7601331 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">エコノグラジエントポンプ</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">7319001</a> </td><td>低圧タンパク質精製のための勾配ポンプは、チューブと継手キットが含まれています</td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experionシステム</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td>bio-rad.com/prd/en/US/adirect/biorad？CMD = BRCatgProductDetail＆ProductID =に224301 &quot;ターゲット=&quot; _blank &quot;&gt; 7007001 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">Experion Pro260分析キット</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">バイオ·ラッド</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">7007102</a> </td><td></td></tr><tr><td>ハイトラップHICカラム選択キット</td><td> GEヘルスケア</td><td> 28-4110-07 </td><td>スカウトのためにHICのメディアの7×1ミリリットルプレパックドカラムが含まれています</td></tr><tr><td> GEヘルスケア·ツー·Bio-Rad社のカラムフィッティング</td><td> GEヘルスケア</td><td> 18111251と18111257 </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

精製タンパク質（例えば、ゲル濾過、アフィニティー、イオン交換）用の他のクロマトグラフィー法とは対照的に、疎水性相互作用クロマトグラフィー（HIC）は、一般に最も適したクロマトグラフ媒体を選択するために実験的な決定を（スクリーニングまたは &quot;スカウト&quot;と呼ばれる）が必要です与えられたタンパク質1を精製する 。ここで紹介する方法は、タンパク質精製に使用する最適なHICメディアのスカウトへの自動アプローチを説明します。 HICは、疎水性の差に基づいて粗ライセートからタンパク質などの生体分子を分離しています。アフィニティークロマトグラフィー（AC）とイオン交換クロマトグラフィー（IEX）と同様に、HICは、クロマトグラフィープロセスの進行に応じて、目的のタンパク質を濃縮することができます。最高のHICによる精製に適したタンパク質は、疎水性表面領域を有するものが含まれており、2 Mの弾薬を超える濃度の塩への暴露に耐えることができるnium硫酸塩（（NH 4）2 SO 4）。 HICは、多くの場合、アフィニティタグを欠いているタンパク質の精製法として選択し、ACのため、適さない、とIEXは、十分な精製を提供するために失敗したときにされています。タンパク質表面の疎水性部分は、一時的に不活性、不動のマトリックスに結合された非極性リガンドに結合する。タンパク質とリガンドとの相互作用は、タンパク質-リガンド相互作用を強化し、不動のタンパク質（すなわち、列の内側に結合した2）を作る高イオン濃度で、クロマトグラフィーカラムを通過する緩衝液の塩濃度に大きく依存します。塩濃度が低下すると、タンパク質 - リガンド相互作用は消費電力、タンパク質は再び携帯電話になり、カラムから溶出する。 HIC複数の媒体がプリパックドカラムで市販されている、複数の疎水リガンド（例えばオクチルs-ブチル、ブチルおよびフェニル）のそれぞれ含むものには規格のアガロースビーズに濃度を変化させて架橋タイプIFIC直径3。 HICメディアが将来、より徹底的な最適化の実験やタンパク質精製の ​​ために採用すべきかを決定するための効率的なアプローチは、4を実行するためにスカウト自動化された列ができます。 ここで精製されている特定のタンパク質は、組換え緑色蛍光タンパク質（GFP）であるが、アプローチは、1つまたは複数の疎水性表面領域と他のタンパク質を精製するために適合させることができる。 GFPは紫外線を5にさらされたとき、その安定性、397 nmでのユニークな吸光度のピークと蛍光による有用なモデルタンパク質として機能します。野生型GFPを含む細菌ライセートは、高塩緩衝液中に調製Bio-Rad社DuoFlow媒体圧力液体クロマトグラフィーシステムにロードされ、異なるHICメディアを含むハイトラップHICカラムに吸着させた。タンパク質はカラムから溶出し、内の行と、実行後の検出法によって分析した。バッファ·ブレンディング、動的なサンプルループ注入、シーケンシャルCOLUMN選択、マルチ波長分析、分割割合溶出コレクションは、実験的アプローチのシステムおよび再現性の機能を増加した。

Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

タンパク質精製のプロセスで使用される適切な疎水性相互作用クロマトグラフィー（HIC）メディアを識別するための自​​動化された方法が示されています。メソッドは、自動化されたバッファ·ブレンディング、動的なサンプルループ注入、順次列の選択、マルチ波長分析、分割割合溶出コレクションなどの中圧液体クロマトグラフィーシステムを利用しています。

タンパク質の精製で使用するための自​​動化された疎水性相互作用クロマトグラフィーカラムの選択

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

automated-hydrophobic-interaction-chromatography-column-selection-for

Research

JoVE Journal

Biology

43.1K ビュー.  Seattle University. タンパク質精製のプロセスで使用される適切な疎水性相互作用クロマトグラフィー（HIC）メディアを識別するための自​​動化された方法が示されています。メソッドは、自動化されたバッファ·ブレンディング、動的なサンプルループ注入、順次列の選択、マルチ波長分析、分割割合溶出コレクションなどの中圧液体クロマトグラフィーシステムを利用...

ビデオ: Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

生物学

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

ヘリオス遺伝子銃を用いたタマネギの細胞における蛋白質 - 蛋白質相互作用のための二分子蛍光相補（BiFC）アッセイ

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

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

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

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

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

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

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

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104の精製、タンパク質Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

組換えタンパク質ベイトを用いてファージディスプレイおよび親和性選択のためのプロトコル

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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

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

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

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

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

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

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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

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

ペプチド配列の使用タンパク質間相互作用部位を同定

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

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 ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

ベンサミアーナ タバコ葉蛋白質蛋白質の相互作用ダイナミクスの一過性の決定のための試金補完ルシフェラーゼ イメージング

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

タンパク質の精製で使用するための自​​動化された疎水性相互作用クロマトグラフィーカラムの選択

タンパク質の精製で使用するための自動化された疎水性相互作用クロマトグラフィーカラムの選択