AFは欧州共同体の第7次フレームワーク·プログラムの下での欧州研究評議会から始まる助成金によってサポートされていました（FP7 / 2007年から2013年）/ ERCグラント契約がn 203413を°、複雑なsystems.HAバイオハイブリッドとAIのためのミネルヴァセンターがサポートされていますダリアとダン·メイダンフェローシップによるエルサレムのヘブライ大学で高度な学位学生のため。

1.ペプチドアレイの設計<ol><li>一部は10〜20残基の重複ペプチドへの標的タンパク質の配列を分割する。具体的な実験と行うラボのリソースにオーバーラップの量を変化させるが、原理的にはより長いオーバーラップがより良い。ペプチドを設計する場合の相互作用を担当することができるタンパク質の二次構造要素を知ら考慮する。 </li><li>商業ベンダーを通じて設計されたペプチドアレイを注文。ここでは、共有結合C末端を通じてアレイに結合したペプチドを使用しています。 </li></ol> 2.非特異的結合をブロック<ol><li> NaClで0.05％のTween 20（TBST / PBST）を含む50mMトリスまたはリン酸緩衝液を作り、（正確なイオン強度を知るために）のHClまたはNaOHの測定量で所望のpHに調整し、所望のイオン強度。ここでは、7.5のpHおよび150mMのイオン強度を使用しています。 </li><li>ブロッキング溶液を作っ2.5％（w / vの）をTBST / PBST中脱脂粉乳。 </li><li>非特異的結合を防ぐために、ブロッキング溶液5mlにアレイを浸す。室温または一晩シェーカー上で4ºCで2〜4時間の配列をインキュベートする。 </li></ol> 3.タンパク質とインキュベート<ol><li>室温でシェーカー上で5分間5ミリリットルTBST / PBSTで二回、次に30秒間ブロッキング溶液と5mlで最初の配列を洗ってください。 </li><li> （w / v）の非特異的結合を防ぐために、脱脂粉乳、2.5％を含有するHisタグ化タンパク質溶液5mlで洗浄し、アレイをインキュベートする。ここでは、説明したブロッキング溶液中に溶解したタンパク質溶液の4.5μM（STIL 500〜650）を使用します。 NOTE：通常5-10μMタンパク質は（スクリーニングに使用されるが、タンパク質濃度は、結合親和性及びアレイで結合したペプチドの局所濃度に依存して、2-3μMでさえ低くすることができる効率合成）。バッファ内のタンパク質変化するが、上記と同じブロッキング溶液を用いてタンパク質を希釈することが一般的であることができ溶解する。 </li><li>室温または一晩、4ºCで3-8時間、タンパク質溶液中の配列をインキュベートする。 </li></ol> 4.抗体とインキュベート<ol><li> 5ミリリットルTBST / PBSTで配列を3回洗う。最初の洗浄は、室温でシェーカー上に2つの5分間の洗浄、続いて30秒間である。 </li><li>室温でシェーカー上で1時間、ブロッキング溶液と同じ濃度で同じ成分を含有するインキュベーション緩衝液中で：希釈したHRP結合抗体（1,500 1）5mlで洗浄し、アレイをインキュベートする。抗体が標的タンパク質またはタグのいずれかを結合することができる。 </li><li>セントせずに同じプロトコルを繰り返すことにより、アレイは、標的タンパク質（ 例えば 、オリゴマー化のための研究）からのペプチドが含まれている場合は特に、アレイ上のペプチドと抗体との相互作用を試験する対照実験を行うp 3が、タンパク質と共にインキュベートする。 </li><li> 5ミリリットルTBST / PBSTで配列を3回洗う。最初の洗浄は、室温でシェーカー上に2つの5分間の洗浄、続いて30秒間である。 </li></ol> 5.アレイを読む<ol><li> ECLウエスタンブロッティング基質キットで化学発光開発を行う。 </li><li>発光画像解析装置で検出を行う。 </li><li>結果を分析します。アレイ上の両方の重複が同じシグナルを示すので、結果は信頼性があることを確認してください。タンパク質パートナーの構造がわかっている場合、結合ペプチドのための構造上の検索および結合部位を探します。遠くの順序で並んでいても、ペプチドは、三次構造に一緒に近くなるとの結合部位を作成することができます。 </li></ol>

<ol>
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B., Buus, S., Schafer-Nielsen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Mapping+of+Linear+Antibody+Epitopes+in+Human+Serum+Albumin+Using+High-Density+Peptide+Arrays.">Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays.</a> PLoS ONE. 8 (7), (2013).</li><li>Uecker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+substrate+peptide+for+the+FLT3+receptor+tyrosine+kinase.">A substrate peptide for the FLT3 receptor tyrosine kinase.</a> British Journal of Haematology. 144 (1), 127-130 (2009).</li><li>Gabizon, R., Faust, O., Benyamini, H., Nir, S., Loyter, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relationship+studies+using+peptide+arrays:+the+example+of+HIV-1+Rev-integrase+interaction.">Structure-activity relationship studies using peptide arrays: the example of HIV-1 Rev-integrase interaction.</a> Med. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SIL+gene+is+required+for+mouse+embryonic+axial+development+and+left-right+specification.">The SIL gene is required for mouse embryonic axial development and left-right specification.</a> Nature. 399 (6737), 691-694 (1999).</li><li>Izraeli, S., Colaizzo-Anas, T., Bertness, V. L., Mani, K., Aplan, P. D., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+SIL+gene+is+correlated+with+growth+induction+and+cellular+proliferation.">Expression of the SIL gene is correlated with growth induction and cellular proliferation.</a> Cell Growth Differ. 8 (11), 1171-1179 (1997).</li><li>Arquint, C., Sonnen, K. F., Stierhof, Y. -. D., Nigg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Cell-cycle-regulated+expression+of+STIL+controls+centriole+number+in+human+cells.">a Cell-cycle-regulated expression of STIL controls centriole number in human cells.</a> Journal of Cell Science. 125 (5), 1342-1352 (2012).</li><li>Tang, C. J., Lin, S. 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+human+microcephaly+protein+STIL+interacts+with+CPAP+and+is+required+for+procentriole+formation.">The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.</a> Embo J. 30 (23), 4790-4804 (2011).</li><li>Vulprecht, J., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIL+is+required+for+centriole+duplication+in+human+cells.">STIL is required for centriole duplication in human cells.</a> Journal of Cell Science. 125 (5), 1353-1362 (2012).</li><li>Campaner, S., Kaldis, P., Izraeli, S., Kirsch, I. 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H., Britan-Rosich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Vif&#8211;A3G+interaction+using+peptide+arrays:+A+basis+for+anti-HIV+lead+peptides.">Mapping the Vif&#8211;A3G interaction using peptide arrays: A basis for anti-HIV lead peptides.</a> Bioorganic &amp; Medicinal Chemistry. 21 (12), 3523-3532 (2013).</li><li>Katz, C., Benyamini, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+interaction+between+the+antiapoptotic+Bcl-2+family+proteins+and+the+proapoptotic+protein+ASPP2.">Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.</a> Proc Natl Acad Sci U S A. 105 (34), 12277-12282 (2008).</li><li>Katz, C., Zaltsman-Amir, Y., Mostizky, Y., Kollet, N., Gross, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Basis+of+the+Interaction+between+Proapoptotic+Truncated+BID+(tBID)+Protein+and+Mitochondrial+Carrier+Homologue+2+(MTCH2)+Protein:+KEY+PLAYERS+IN+MITOCHONDRIAL+DEATH+PATHWAY.">Molecular Basis of the Interaction between Proapoptotic Truncated BID (tBID) Protein and Mitochondrial Carrier Homologue 2 (MTCH2) Protein: KEY PLAYERS IN MITOCHONDRIAL DEATH PATHWAY.</a> Journal of Biological Chemistry. 287 (18), 15016-15023 (2012).</li><li>Rosenberg, M. 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The authors have nothing to disclose.

ペプチドアレイスクリーニングは、そのパートナーで標的タンパク質の結合部位を同定するための優れたツールである。このアッセイは、実行するために迅速かつ容易であり、結果は、1つまたは2つの営業日で得ることができる。ペプチドアレイスクリーニングは汎用性があり、多くの目的に使用することができる。これは、リン酸化などの翻訳後の修飾を特徴付けるし、キナーゼの基質を?...

タンパク質 - タンパク質相互作用は、生細胞内のプロセスの大部分を媒介する。損なわれたタンパク質相互作用は、タンパク質相互作用に重要な薬剤標的を作る疾患をもたらし得る。これは、分子レベルでこれらの相互作用を理解することが非常に重要である。タンパク質相互作用は、細胞性及び生化学的アッセイから定量的な生物物理学的アッセイの範囲の様々な技術を用いて研究されており、これらは、タンパク質ドメイン、またはペプチドと、全長タンパク質のいずれかで実施することができる。ペプチドは、タンパク質相互作用を研究するための優れたツールとして役立つ。ペプチドは容易に合成され、一方、他方の1,2-高スループット様式での複数のタンパク質標的上の特定の相互作用部位に焦点を可能にすることができるからである。ペプチドアレイスクリーニングは、短時間3に多数のパートナーと標的タンパク質との相互作用に関する大量のデータを得るための方法を実行するのは簡単、高速で。タンパク質 - タンパク質相互作用を検出し、分析するための他の生化学的または生物物理学的方法とは異なり、ペプチドアレイスクリーニングは、タンパク質の非常に低い濃度を必要とし、非常に弱い結合を検出することができる。ペプチド配列は、酵素活性6と高スループットを研究する抗体エピトープ5を特徴付ける、そのようなタンパク質-タンパク質または受容体-リガンド相互作用部位4、ホモまたはヘテロオリゴマー化インターフェースのマッピングのようなペプチド-タンパク質相互作用の多くの用途に使用することができる構造-活性関係（SAR）は7を研究。ペプチドアレイのスクリーニングについての詳細なレビューについてはカッツらを参照してください。4 ペプチドアレイのいくつかのタイプは、現在存在している。固体支持体にそれらを取り付ける前に、ペプチドの合成、または固体支持体上に直接ペプチドの合成は主に、SPOT技術の4,8を使用して、：ペプチドアレイを作製するための2つの主要な合成戦略があります。ザ·ペプチドは、9-フルオレニルメチルオキシカルボニル（Fmoc）化学8、通常、固体支持体上で合成される。一般的な合成スキームの中でC末端を介してペプチドのN末端 ​​を介してアタッチメント（ 例えば、JPTのpepstarアレイ9）およびペプチドアタッチメントがある（ 例えば、PEPSCANのpepchipアレイ10、JPTのペプスポットアレイ9とINTAVISのcelluspotアレイ11）4。固体支持体は変わるので、それへのペプチド結合の化学を行いますことができます。システイン-末端ペプチドは、チオール基12を介してスライドガラスに付着させることができる。ペプチドのN末端 ​​に共有結合したアミノ酸のエステル化セルロース膜上のヒドロキシル基に結合することができる8を取り付け。 ここでは、タンパク質 - タンパク質相互作用を研究するための方法として、ペプチドアレイをスクリーニングするための詳細なプロトコールを提示する。私たちが使用する配列は、大規模なAMOを含むマイクロアレイであるCelluspots配列ですスライドガラスでサポートされている小さなセルロース膜上にスポット（最大384スポットの重複）のUNT。これは、低タンパク質のボリュームと抗体と協力し、一回の実験あたりのデータのかなりの量を得ることができます。この配列はまた、低親和性結合の検出を可能にする高いペプチド密度が含まれています。アレイは、正常な細胞増殖13を制御するために非常に重要であるSTIL、CHFRの相互作用をマッピングするために使用した。 2つのタンパク質間の制御されない相互作用が癌の発生につながる可能性がある。この相互作用をマッピングすることによって、私たちは、特定の結合部位を発見し、残基14を結合。これは、このタンパク質 - タンパク質相互作用を阻害する阻害剤の合理的設計のための道を開く。

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:231px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptide array</td>
			<td>INTAVIS Bioanalytical Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">His-probe Antibody (H-3) HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8036 HRP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-ECL Kit</td>
			<td>Biological industries</td>
			<td>20-500-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skim Milk</td>
			<td>Becton, Dickinson and Company</td>
			<td style="width:119px;">232100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LAS-3000 camera&#160;</td>
			<td>FUJI film</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction sites using peptide arrays

タンパク質 - タンパク質相互作用は、生物の生きている細胞および対照恒常性プロセスの大部分を媒介する。損なわれたタンパク質相互作用は、タンパク質相互作用に重要な薬剤標的を作る疾患をもたらし得る。これは、分子レベルでこれらの相互作用を理解することが非常に重要である。タンパク質相互作用は、細胞性及び生化学的アッセイから定量的な生物物理学的アッセイの範囲の様々な技術を用いて研究されており、これらは、タンパク質ドメイン、またはペプチドと、全長タンパク質のいずれかで実施することができる。ペプチドは、ペプチドを容易に合成することができるので、タンパク質相互作用を研究し、特定の相互作用部位に焦点を可能にするための優れたツールを提供。ペプチドアレイは、2つのタンパク質間の相互作用部位の同定、ならびに治療目的のために、標的タンパク質に結合するペプチドのスクリーニングを可能にする。彼らはまた、高スループットのSAR研究を可能にします。結合部位の同定のため、typi校正ペプチドアレイは、通常、部分的に、所望の標的タンパク質の1つまたは複数のパートナータンパク質の完全な配列に由来する10〜20残基の重複ペプチドが含まれています。標的タンパク質を結合するためのアレイをスクリーニングするタンパク質の少量のみを使用して簡単かつ迅速な方法では、パートナータンパク質における結合部位に対応し、結合ペプチドを明らかにする。 この記事では、標的タンパク質とそのパートナーの間の相互作用部位をマッピングするためのペプチド配列をスクリーニングするためのプロトコルを記述します。ペプチドアレイを考慮し、それらの二次構造をとるパートナータンパク質の配列に基づいて設計されている。このプロトコルで使用されるアレイは、INTAVISバイオ分析·インスツルメンツによって調製しCelluspots配列だった。アレイは、非特異的結合した後、勉強タンパク質とインキュベートを防ぐためにブロックされます。抗体を用いた検出は、タンパク質間の特定の相互作用部位に対応する結合ペプチドを明らかにする。

STILは非常に重要な中心体タンパク質である。 19 -これは、正常な細胞分裂および細胞増殖13,15を制御する。 STILいくつかのタンパク質18,20,21と相互作用し、相互作用の大部分は、本質的に無秩序領域（IDR）14は 、その中央部を介して起こる。私たちは、STIL結合タンパク質CHFR由来のペプチドで構成される配列を設計しました。 CHFR応力22を有糸分裂に応?...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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 ...

identifying-protein-protein-interaction-sites-using-peptide-arrays

Research

JoVE Journal

Biology

17.9K ビュー.  The Hebrew University of Jerusalem. 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.

ビデオ: Identifying Protein-protein Interaction Sites Using Peptide Arrays

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

生物学

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 ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

イメージングタンパク質間相互作用 in vivoで</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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 ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

二分子蛍光相補のフローサイトメトリー分析：タンパク質 - タンパク質相互作用を研究するためのハイスループット定量法

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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. ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

SPOTペプチド配列を用いてタンパク質リジンメチルトランスフェラーゼの特異性の解析

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

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 ...