Name: chip methods for dna-protein binding
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 36 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Chromatin Immunoprecipitation- ChIP

クロマチン免疫沈降法(略称:ChIP)は、遺伝子発現を調節するタンパク質-DNA相互作用を研究するための手法です。
真核生物では、DNAはヒストンタンパク質に巻き付けられ、ヌクレオソームとして知られる複合体を形成し、さらにヌクレオソームがクロマチンと呼ばれる構造にグループ化されて、細胞内のDNAの緊密なパッケージングを助けます。
遺伝子発現は、これらの構造をほどいたり引き締めたりするヒストン修飾や、DNA上のシス調節領域に結合するタンパク質によって調節されます。
ChIPでは、クロマチンが分解され、ヒストン修飾または調節タンパク質に結合する抗体を使用して、関連するDNAを持つ標的分子を単離します。
このプロセスの最初のステップは、ホルムアルデヒドなどの架橋剤を使用して、タンパク質をDNAに架橋することです。これにより、タンパク質はDNA上に固定化され、タンパク質の結合部位をマーキングします。架橋後、クロマチンは機械的に100〜200塩基対の範囲の短い断片にせん断されます。このプロセスはX-ChIPとして知られています。
別の方法はN-ChIPで、ヌクレアーゼは事前に架橋することなく、クロマチンからDNAを直接短い断片に消化します。
免疫沈降は、剪断または消化されたDNAを含む溶液中の調節タンパク質を標的とする抗体を導入することによって行われます。これらの抗体は、選択的単離に役立つ薬剤と関連しています。
一般的な方法の1つは、抗体を磁気ビーズに結合することです。磁石を使用して、結合した分子とともに抗体を分離します。
複合体をすすぎ、緩く結合した汚染物質を洗い流します。標的分子は、SDSなどの界面活性剤を使用して剥離されます。
X-ChiPの場合、架橋は高温の助けを借りて逆転し、関連するレギュレーターまたはヒストンはプロテアーゼの助けを借りて分解され、DNAが残ります。 
関連するDNAのシーケンシングは、目的のタンパク質が結合したシス制御配列、または特定のヒストン修飾によって発現が調節される遺伝子を特定するのに役立ちます。

<ol>
	<li>Gade, P., & Kalvakolanu, D. V. (2012).転写因子活性を解析するためのツールとしてのクロマチン免疫沈降アッセイ。分子生物学の方法(クリフトン、ニュージャージー州)、809、85-104 https://doi.org/10.1007/978-1-61779-376-9_6。</li>
	<li>ネルソン、JD、デニセンコ、O.、およびBomsztyk、K.(2006)。高速クロマチン免疫沈降(ChIP)法のプロトコル。ネイチャープロトコル、1(1)、179-185。土井:10.1038/nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J., & Singal, R. (2004).クロマチン免疫沈降アッセイ。バイオテクノロジー, 37(6), 961-969.</li>
</ol>

クロマチン免疫沈降 - chip

クロマチン免疫沈降(ChIP)は、目的の転写因子またはヒストンタンパク質に結合するDNA上の部位を特定するために使用される抗体ベースの技術です。また、アセチル化、リン酸化、メチル化などのヒストン修飾の種類を決定するのにも役立ちます。

ChIPの種類

ChIPは、X-ChIPとN-ChIPの2つのタイプに分類できます。X-ChIPには、ヒストンおよび調節タンパク質のDNAへのin vivo架橋、超音波処理によるDNAの断片化、および特異的抗体による免疫沈降によるタンパク質 - DNA複合体の単離が含まれます。一方、N-ChIPにはDNAとタンパク質の架橋は含まれず、消化はヌクレアーゼを使用して実行されます。

DNA - タンパク質相互作用の下流解析

目的のDNAを単離した後、PCR、マイクロアレイ、サザンブロットなどの技術を分析に使用できます。あるいは、このDNAは、ChIP-Seqとして知られるディープ シーケンスに使用することもできます。ChIPは、ChIPの原理と染色体立体構造捕捉を組み合わせて、目的のタンパク質を介して媒介される長距離のクロマチン相互作用を検出する技術であるChIA-PETなど、さまざまな分析のために他の方法論を使用して変更できます。enChIPは、CRISPR/Cas9システムを使用して特定のゲノム領域をターゲットにする技術、およびRIP-ChIP/RIP-Seqは、タンパク質とRNAの相互作用を分析するために使用されるChIPの改良版です。

N-ChIPとX-ChIPの比較

N-ChIPとX-ChIPには、それぞれ長所と短所があります。N-ChIPは、より強力な抗体結合と効率的かつ高度に特異的な免疫沈降をもたらします。ただし、N-ChIPは、転写因子が処理中に分離される可能性があるため、ヒストンなどの強く結合したタンパク質の場合にのみ適しています。さらに、ヌクレアーゼで消化されたクロマチンのすべてが可溶化されるわけではないため、サンプルの特定の画分が失われることになります。X-ChIPは、架橋ステップによりDNAにしっかりと結合していない転写因子を研究するための優れた方法論です。 X-ChIP アッセイはN-ChIPよりも感度が高く、必要なサンプルと抗体の量が少なくなります。Xチップの欠点としては、過剰な架橋により断片化が困難になる可能性や、一時的なDNAタンパク質相互作用の架橋により偽陽性が発生する可能性があることが挙げられます。

クロマチン免疫沈降 - ChIP

クロマチン免疫沈降法(ChIP)は、目的の転写因子に結合するDNA上の部位を同定するために使用される抗体ベースの技術です。

Education

JoVE Core

Molecular Biology

分子生物学

Unit 2

ユニット2

Analyzing Gene Expression and Function

遺伝子発現と機能の解析

KEY TERMS AND CONCEPTS

主要用語と概念

chip methods for dna-protein binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation.

ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes are then isolated by immunoprecipitation with specific antibodies. N-ChIP does not use cross-linking. Instead, it uses nucleases to digest the chromatin.

After the DNA of interest is isolated, it can be analyzed in several ways. PCR, microarrays, and Southern blot can be used to study the captured DNA. The sample can also be sent for deep sequencing, which is called ChIP-Seq.

ChIP can also be adapted for other kinds of analysis. ChIA-PET combines ChIP with chromosome conformation capture to detect long-range chromatin interactions that are mediated by a protein of interest. enChIP uses the CRISPR/Cas9 system to target specific genomic regions. RIP-ChIP and RIP-Seq are used to study protein-RNA interactions.

N-ChIP and X-ChIP each have strengths and limits. N-ChIP gives stronger antibody binding and efficient, highly specific immunoprecipitation. It works best with tightly bound proteins such as histones, but transcription factors may be lost during processing. Some nuclease-digested chromatin may also remain insoluble, so parts of the sample can be missed.

X-ChIP is useful for studying transcription factors that do not bind tightly to DNA because it includes a cross-linking step. It is also more sensitive than N-ChIP and uses less sample and fewer antibodies. Its drawbacks include possible problems with fragmentation if cross-linking is too strong and false positives from transient DNA-protein interactions.

ChIP Methods for DNA-Protein Binding

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chromatin immunoprecipitation- chip

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation. ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes...

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key terms and concepts

Explore how ChIP detects DNA-protein binding, compares X-ChIP and N-ChIP, and shows how captured DNA can be analyzed by PCR, sequencing, and more.

ChIP Methods for DNA-Protein Binding | JoVE K12

molecular biology

JoVE Core 分子生物学は、明確で簡潔なアニメーション ビデオ レッスンを通じて細胞の分子プロセスを紹介します。さらに、科学者の実際のビデオでは、これらの概念の応用が現代の研究室での実際の実験を通じて実証されています。

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BIO1.ETS2.3

ChIP Methods for DNA-Protein Binding

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