Name: a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis
Uploaded: 2017-01-24T12:30:00
Description: Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), the Takeda Science Foundation (HT), and by a Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) to HT.

1.トランスフェクション<ol><li> 100 mmの培養プレートでHEK293細胞を播種（2×10 6細胞/皿）。培養10％ウシ胎児血清、100μg/ mlのストレプトマイシン、および100単位/ mlのペニシリンを補充したダルベッコ改変イーグル培地中の細胞。 5％CO 2、37℃の加湿インキュベーター中で細胞をインキュベートします。 </li><li>翌日、製造業者の指示11に従ってトランスフェク?...

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効率的なトランスフェクションは、このプロトコルで成功した結果を得ることが不可欠です。したがって、我々は免疫沈降FLAGタグ付きタンパク質レベルを決定するために、ウェスタンブロット分析を使用することをお勧めします。このステップでは、IPが正常に実行されたこと、また、興味のあるタンパク質が適切に過剰発現していることを確認し、することができます。 FLAG標識タンパク?...

タンパク質 - タンパク質相互作用は、多くの生物学的機能において重要な役割を果たしています。このように、これらの相互作用は、シグナル伝達に関与しています。細胞膜を横切るタンパク質輸送;細胞代謝; DNA複製、DNA損傷修復、組換え、および転写1、2、3、4を含む、いくつかの核プロセス。これらの相互作用に関与するタンパク質を同定することは、これらの細胞プロセスの理解を進めることが重要です。 免疫沈降（IP）は、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る検証技法です。共免疫沈降したタンパク質の同定を容易にするために、質量分析がしばしば利用されて5、6、7、8、9。抗体とタンパク質複合体の既知のメンバーを標的とすることにより、タンパク質複合体を単離するために、続いて質量分析を介して、その未知の成分を同定することが可能です。 ARIP4（アンドロゲン受容体相互作用タンパク質4）、転写補調節因子は、文脈に依存した方法9、10でその標的プロモーターを活性化または抑制するために、核内受容体タンパク質と相互作用します。より良いこれらの核因子を支配する転写調節機構を理解するために、我々は、精製し、LC-MS / MSシステムを使用してARIP4相互作用するタンパク質を同定するための包括的方法を記載しています。

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			<td height="81" style="height:81px;width:216px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:71px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11668019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Protease Inhibitor Cocktail (EDTA free) (100x)</td>
			<td style="width:71px;">nacalai tesque</td>
			<td style="width:119px;">03969-21</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ANTI-FLAG M2 Affinity Gel</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Micro Bio-Spin Columns</td>
			<td style="width:71px;">BIO-RAD</td>
			<td style="width:119px;">732-6204</td>
			<td></td>
		</tr>
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a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

私たちは、160 KDaの周りに、強いARIP4信号を識別し、同様にモックサンプル制御および他のいくつかの未知のタンパク質（ 図1）のもの。 LC-MS / MS分析は、FLAGビーズ（ 表1）の画分内ARIP4複合ペプチド及び潜在的なペプチド補因子の両方を同定しました。 P62（Sequestosome1）、既知ARIP4補因子11は、このシステム11?...

Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

我々は、LC-MS / MSシステムを使用して共調節相互作用タンパク質の精製のための方法を確立しました。

LC-MS / MS分析を用いた核補因子と相互作用するタンパク質のハイスループット同定のためのタンパク質調製方法

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

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本質的に無秩序なタンパク質の複数のリン酸化の同定のための核磁気共鳴分光法

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成熟米の横断断面(オリザ・サティバ L.ピペットチップを固定化支援として用いた走査電子顕微鏡イメージング用カーネル

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Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

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Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

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Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...