当研究室での作業はNIH、食と農のUSDA国立研究所、NSF、BARD、DOE、及びBSFか​​らVCへの補助金によってサポートされています

1。タンパク質の発現と抽出<ol><li>差彼らの検出をテストするタンパク質にタグを付ける。大きいサイズ（例えば、GST）のタグ付き膜（ProIM）上に固定化するタンパク質にラベルを付け、そして融合していないタグが固定されたネガティブコントロール（ProIMnc）として使用することができます。大規模または小規模なタグのいずれかを持つ水溶性プローブ​​（ProSOL）として使用するタンパク質にラベルを付けます。適切な陰性対照可溶性タンパク質（ProSOLnc）に同じタグを融合し、検出された結合の特異性を確認するために、相互作用のアッセイに含まれています。 </li><li>すなわち、E.、タグ付きタンパク質を発現する蛋白質の発現系を選択します。 大腸菌 、バキュロウイルス、等、タンパク質の潜在的な翻訳後修飾と収量のための要件に応じて。 </li><li>プロテイナーゼ阻害剤カクテルを含むSDS - PAGEローディングバッファー（20％グリセロール、4％SDS、20mMトリス- HCl、pH6.8）を用いて選択肢の生物（ステップ1.2）からProIMとProIM ncを抽出する。 15分間室温で細胞懸濁液のまま、2 - メルカプトエタノールおよび5分間沸騰の0.5％を追加。 </li><li>標準プロトコル16を使用して選択肢の生物（ステップ1.2）からProSOLとProSOL ncを抽出する。これらのタンパク質は、ステップ4.1で使用される結合バッファー（140mMのNaCl、10mMのトリス塩酸pH7.4の、2mMのEDTA、2mMのDTT、1％BSA、0.1％のTween 20）で容易に溶解する必要があります。 </li><li>このようなバイオラッドのプロテインアッセイキットなどの標準的な方法を用いて組換えタンパク質の濃度を決定する。バイオラッドのプロテインアッセイキットなど。粗抽出物ではなく、タンパク質の精製は、アッセイに使用されている場合は、クマシーブリリアントブルーR - 250と、既知の使用に染色したSDS -ポリアクリルアミドゲル上の対応するバンドの密度測定をスキャンすることにより、この抽出物中の目的のタンパク質の濃度を推定リファレンスとしてBSAの濃度。 </li></ol> 2。メンブレン上のProIMとProIMncの固定化<ol><li>抽出物の2セット標準プロトコル16に記載のSDS -ポリアクリルアミドゲル上ProIMとProIM NC1μgを含む各を解決します。 </li><li>電気泳動後、転送バッファの100ml（60 mMグリシン、10mMのトリス、0.0006パーセントSDS、20％メタノール）で、転送のゲルを置き、室温で20分間穏やかに撹拌しながらインキュベートする。 </li><li>標準プロトコル16に記載のニトロセルロース膜にゲルに含まれるタンパク質をElectrotransfer。 </li></ol> 3。膜結合タンパク質の再折りたたみ<ol><li> electrotransferした後、バッファー15ml中に15分間メンブレンをインキュベート（30 mMトリス- HCl pH7.4の、0.05％ツイーン20）残SDSを除去するために穏やかに振とうさせながら。 </li><li>慎重に排出した後、変性バッファー（7 Mグアニジン塩酸塩、2mMのEDTA、50mMのDTT、50mMのTris - HCl pH8.3）を25mlのに膜を転送する。と穏やかに撹拌しながら室温で2時間インキュベートする。 （注：変性バッファー中でインキュベートしたとき、ニトロセルロース膜が不透明になる）。と穏やかに撹拌しながら室温で2時間インキュベートする。 </li><li> 25 TBSの液（10mMのTris - HCl、150mMのNaCl、pH7.4）に、5分間穏やかに振とうさせながらインキュベートする膜を転送する（注：このステップでは、膜は元の白い色を取り戻す）。 </li><li>結合バッファーの25mlにメンブレンを移し（ステップ1.2参照）、4℃でインキュベート一晩穏やかに撹拌しながらC。 </li></ol> 4。 ProSOLでProIMをプロービング<ol><li> 2つのストリップ、両方を含むProIMとProIM NCに膜を切り取ります。 </li><li>新鮮な結合緩衝液10mlでProSOLまたはProSOLncどちら1-10μgの準備を希釈することによりProSOLとProSOLncハイブリダイゼーション溶液を加えます。 ProSOLまたはProSOLncハイブリダイゼーション溶液中にそれぞれの膜を転送し、穏やかに撹拌しながら室温で1.5時間インキュベートする。 </li><li>ハイブリダイゼーション溶液から膜を削除し、TBSで各15分を3回すすいでください。 </li></ol> 5。免疫ブロッティングによりタンパク質間相互作用の可視化<ol><li>室温で1時間TBSTでは2.5％スキムミルク（10mMトリス- HCl、140mMのNaCl、0.05％ツイーン20、pH7.4）でメンブレンをブロックする。メーカーが推奨する濃度でTBSTで0.5％スキムミルクで一次抗体（抗strepIIポリクローナルウサギ抗体）を希釈します。 </li><li>抗体溶液でブロックされた膜を配置し、4℃、室温で1時間または一晩インキュベート℃で穏やかに振とうさせながら。 </li><li> 15分間、2回穏やかに撹拌しながら室温で5分間、20mlのTBSTで膜を洗浄します。 </li><li>メーカーが推奨する濃度でTBSTで0.5％スキムミルクでホースラディッシュペルオキシダーゼ（HRP）を結合させた二次抗体（抗ウサギIgG抗体）希釈します。二次抗体のメンブレンを置きソリューションと穏やかに撹拌しながら室温で1時間インキュベートする。 </li><li> 15分間、2回穏やかに撹拌しながら室温で5分間、20mlのTBSTで膜を洗浄します。最終的には、TBSで洗浄後、HRPの化学発光基質（例えば、ミリポアイモビロン西部化学発光HRP基質）を用いて、タンパク質 - タンパク質相互作用を可視化する。 </li><li>ステップ5.4から5.1で説明したように、適切な二次抗体が続くProIMに融合されたタグ用の一次抗体と同じ膜を、プローブ。視覚化ProIMとProIMncは、タンパク質の膜のオーバーレイアッセイ（ステップ5.5）で得られたバンドのアイデンティティを検証するためにステップ5.5で説明。この段階で得られた信号は、ステップ5.5から得られる残差信号よりもはるかに強力であるため、ほとんどの場合、ストリッピングは、必要ありません。 </li></ol> 6。代表的な結果： ANK - MPとの対話は、タバコ表皮細胞（図1A）にBiFCで観察された。 MPは、細菌や植物において発現する高度に不溶性のタンパク質であるため、タンパク質の膜のオーバーレイアッセイは 、in vitro（図1B） におけるこの相互作用を検証するために採択されました。 GST - MP（ProIM）または非融合GST（ProIM NC）1μgを含むタンパク質抽出物は、ニトロセルロース膜にelectrotransfer続いて、SDS -ポリアクリルアミドゲル電気泳動により分離した。これらProIMsは、水溶性ANK - strepII（ProSOL）、GST - MPが、融合していないではないGSTでプローブ結合展示されたとき（図1B、レーン1、2、レーン5、6に比較して）。また、ProIMsの同じセットが無関係ProSOLnc、すなわち、strepII（NADH3 - strepII）タグ付けされたシロイヌナズナ細胞質 NADHキナーゼでプローブしたときに、バインディングが観察されていない、さらにANK - MPの相互作用（図1B、レーンの特異性を実証3、4）。 <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="図1"/> 図1。in vivoおよび in vitro における TMV MPのタバコANKの特異的結合。 BiFCによって検出されたとしてタバコ表皮細胞を生体内に（A）ANK - MPとの対話。 MPとANKは、C末端とそれぞれYFPのN末端半分、に融合されたとき強いYFP信号が再構成され、それらのコードする遺伝子のmicrobombardment以下のタバコの表皮に共発現させた。プラスモデスム15の診断である細胞の周囲、で涙点に蓄積されたこのBiFC信号。 in vitroでの （B）ANK - MPの相互作用などの蛋白質の膜のオーバーレイアッセイによって検出。 GST - MP（ProIM）または非融合GST（ProIMnc）1μgを含むタンパク質抽出物は、ニトロセルロース膜上にelectrotransfer続いて15％SDS -ポリアクリルアミドゲル上で分離した。 GST - MPProIMとGSTProIMncはANK - strepII（ProSOL）0.5μg/ mlのと共にインキュベートし、ANKの結合は、への結合抗ウサギIgG + M二次抗体に続いて、抗strepIIウサギポリクローナル抗体と膜をプローブによって検出されたHRP（レーン1および2）。 GST - MPProIMもGSTProIMncどちらもstrepIIタグ（ProSOLnc、レーン3および4）に融合された無関係なタンパク質、 シロイヌナズナ細胞質NADHキナーゼと相互作用する。このアッセイにおいて観察されたバンドのアイデンティティは、抗GST抗体（レーン5および6）を持つ膜をプロービングにより確認された。 GST - MPおよびGST続きですタンパク質抽出物に含まれている正体不明の蛋白質が重要性を実証、ANK - strepIIと反応しながら膜を緩衝液で洗浄されることなく、変性バッファーで処理した場合、ANKにGST - MPの結合は、失われステップ3.1の前に、変性過程（レーン7および8）。

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利害の衝突は宣言されません。

このアプローチは、タンパク質の組み合わせの間のタンパク質-タンパク質相互作用をテストするのに適している、タンパク質の際に少なくとも一つは、結合バッファーに容易に可溶であり、成功したタンパク質17,18の他の組み合わせに適用した。これらの条件下で両方に不溶であるタンパク質の間iInteractionsは、このプロトコルでテストすることはできません。 ?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>タンパク質アッセイキット</td><td> Bio - Rad社</td><td> 500-0001 </td></tr><tr><td>プロテイナーゼ阻害剤カクテル</td><td> SIGMA </td><td> S8820 </td></tr><tr><td>ミニプロティアンシステム</td><td> BIO - RAD </td><td> 165-8000 </td></tr><tr><td>セミドライウェスタンブロッティングSD electrotransferシステム</td><td> BIO - RAD </td><td> 170-3940 </td></tr><tr><td>西洋ワサビペルオキシダーゼと結合抗ウサギIgG抗体</td><td> GenScript </td><td> A00098 </td></tr><tr><td>抗GSTウサギポリクローナル抗体</td><td> GenScript </td><td> A00097 </td></tr><tr><td>アンチstrepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> BioTrace、NTニトロセルロース転写膜</td><td>ポールサイエンティフィック</td><td> 27377-000 </td></tr><tr><td>イモビロン西部化学発光HRP基質</td><td>ミリポア</td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

異なるタンパク質間の相互作用を検証することは、分子レベルでそれらの生物学的機能の研究に不可欠です。そこに結合タンパク質を評価するためのin vitroおよび in vivo の両方でいくつかの方法では、、であり、そしてお互いの欠点を補完少なくとも二つの方法は信頼性の高い洞察を得るために行われるべきである。 in vivoアッセイの場合は、二分子蛍光相補性（BiFC）アッセイは、生きた細胞内のタンパク質-タンパク質相互作用を検出するだけでなく、相互作用するタンパク質の1,2の細胞内局在を識別できる、最も人気があり、最も侵襲的なアプローチを表しています。このアッセイでは、非蛍光性のN -およびGFPまたはその変異体のC末端の半分がテストされたタンパク質に融合させ、そして2つの融合タンパク質が原因でテストタンパク質&quot;相互作用するために一緒に持って来られる時、蛍光シグナルが3-6を再構成する。その信号は、落射蛍光または共焦点顕微鏡によって容易に検出可能であるため、BiFCは、生きた細胞3のタンパク質間相互作用について研究するための細胞生物学者の間で選択の強力なツールとして登場しました。このアッセイは、しかし、時には偽陽性の結果を生成することができます。例えば、蛍光シグナルは限りお互いから7 nmが小さいため細胞内コンパートメントに梱包閉じるには、むしろそのため、特定の相互作用7のように配置された2つのGFPフラグメントによって再構成することができる。 これらの制限のため、生細胞イメージング技術から得られた結果は、タンパク質相互作用を検出するための別の原理に基づいて、独立したアプローチによって確認されるべきである。共免疫沈降法（共同IP）またはグルタチオントランスフェラーゼ（GST）プルダウンアッセイ一般的にin vitroでのタンパク質-タンパク質相互作用を分析するために使用されているような代替方法を表しています。しかし、これらのアッセイIIN、しかし、テストしたタンパク質は、結合反応のためにsupportsusedバッファーに容易に溶解する必要があります。したがって、不溶性のタンパク質が関与する特異的な相互作用は、これらの手法によって評価することができます。 ここで、我々はこの困難を回避する有効な蛋白質の膜のオーバーレイ結合アッセイ、プロトコルを示しています。タンパク質の一つが細胞膜のマトリックス上に固定化されているため、この手法では、水溶性と不溶性のタンパク質間の相互作用を確実にテストすることができます。このメソッドは、そのようなBiFCなどのin vivo実験、 のとの組み合わせで、水溶性と不溶性のタンパク質の間で忠実に相互作用を調査し、特徴付けるために信頼性の高いアプローチを提供します。この記事では、ウイルスの細胞間輸送8-14、および最近同定された植物細胞の相互作用、タバコアンキリン反復を含むタンパク質（ANK中に複数の機能を発揮するタバコモザイクウイルス（TMV）移行タンパク質（MP）、の間の結合）15は 、このテクニックを使用して示されています。

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

テストのタンパク質 - タンパク質相互作用は、タンパク質の機能の解離のために不可欠である。ここでは、導入<em&gt; in vitroで</em可溶性タンパク質と膜固定化タンパク質を調べるために&gt;タンパク質 - タンパク質結合アッセイ。このアッセイは、不溶性タンパク質と溶液中のタンパク質間の相互作用をテストするために信頼性の高い方法を提供する。

タンパク質のメンブレンオーバーレイアッセイ：可溶性および不溶性タンパク質間の相互作用をテストするためのプロトコル in vitroで</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

protein-membrane-overlay-assay-protocol-to-test-interaction-between

Research

JoVE Journal

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

22.0K ビュー.  State University of New York. テストのタンパク質 - タンパク質相互作用は、タンパク質の機能の解離のために不可欠である。ここでは、導入 in vitroでタンパク質 - タンパク質結合アッセイ。このアッセイは、不溶性タンパク質と溶液中のタンパク質間の相互作用をテストするために信頼性の高い方法を提供する。

ビデオ: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

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

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 ...

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 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

脂質中間相における膜タンパク質のハイスループット結晶化のためのロボットの使用

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

タンパク質アルギニンメチル化を研究するためのin vitroでメチル化アッセイで

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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 ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

BIFC-FRET-FLIMアッセイによるMADS型転写因子の2つのモノマーとカルシウムセンサタンパク質の三者間相互作用の測定

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

大腸菌由来の組換えタンパク質充填小胞の作製とイメージング

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

著者スポットライト:組換えタンパク質の生産 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.