我々は、このビデオと記事を批評への参加のためにブラウンとラムラボのメンバーに感謝したい。この作品は、健康研究と健康研究のためのマイケルスミス財団のためのカナダの協会からの資金によって支えられている。

DNAの抽出および検体の調製異なるさまざまなサンプル（培養細胞、新鮮なだけでなく、ホルマリン固定パラフィン包埋組織として冷凍）からDNAをMeDIPに使用することができます。このようなヒストンなどの関連蛋白質することなく、高度に精製されたDNAを使用することが重要です。それはDNAの定量と抗​​体の結合の両方を妨げることができるように、試料からできるだけ多くのRNAを除去することも重要です。 MeDIPのために使用されるDNAの量は200 ngから利用可能なDNAの量に応じて、1μgの範囲で指定できます。このプロトコルを示すために、1μgのDNAが使用されます。以下のプロトコールは、培養細胞から高品質の二本鎖DNAを提供します。他のプロトコルは、他の種類のサンプルからDNAの抽出に使用されるべきである。 <ol><li> 1.7ミリリットルエッペンドルフチュー​​ブ中で細胞ペレットに消化緩衝液400μlを添加します。 </li><li>チューブにプロテイナーゼKの100μgを追加し、50℃一晩インキュベート℃、 </li><li> 500μlのフェノールのpH 7を追加し、反転によって穏やかに混和する。 </li><li>室温で10分間13,000 gでスピン。 </li><li>新しいチューブに水（上部）分数を取り外します。 </li><li>繰り返します一度3〜5を繰り返します。 </li><li> 500μlの1時01フェノール/クロロホルムpHが7を追加し、反転によって穏やかに混和する。 </li><li>室温で10分間13,000 gでスピン。 </li><li>新しいチューブに水（上部）分数を取り外します。 </li><li> RNase Aを40μgを追加し、37℃で1時間のインキュベート℃に</li><li> 500μlのフェノールのpH 7を追加し、反転によって穏やかに混和する。 </li><li>室温で10分間13,000 gでスピン。 </li><li>新しいチューブに水（上部）分数を取り外します。 </li><li>繰り返しは、かつて11〜13を繰り返します。 </li><li> 500μlの1時01フェノール/クロロホルムpHが7を追加し、反転によって穏やかに混和する。 </li><li>室温で10分間13,000 gでスピン。 </li><li>新しいチューブに水（上部）分数を取り外します。 </li><li>の3 M酢酸ナトリウム（40μl）を10分の1の体積を加え、よく混ぜる。 </li><li> 100％エタノールの2倍量（900μl）を追加し、20分、-20 ° Cでよく混ぜ、そして場所。 </li><li> 4℃で20分間13,000 gでスピン℃、 </li><li>エタノールを削除する、パルスのスピン、そして残留エタノールを除去します。 </li><li>洗浄に500μlの冷70％エタノールを加えます。 4℃で20分間13,000 gでスピン℃、 </li><li> 70％エタノール、パルスのスピンを削除し、ピペッティングにより残留エタノールを除去します。 </li><li>空気が残留エタノールの痕跡をすべて削除するには、室温で10分のためのオープンキャップでペレットを乾燥させます。 </li><li> 4℃で一晩滅菌dH2Oの50μlの再懸濁し、DNA℃の</li><li>光度計分光光度計を用いてDNAを定量化する。 260：1.8の280比率が理想的です。 </li><li> 100 bpのラダーをアガロースゲル上でDNAの品質とサイズの範囲を決定します。ゲノムDNAのわずか10 ngのは、SYBRゴールドのようなDNAの少量に非常に敏感な色素を用いて染色を1.7パーセントアガロースゲル上で実行することができます。 </li><li>サンプルごとにシリコン処理したチューブに、滅菌のdH 2 Oで構成されたボリュームの残りの50μlの合計体積で1μgのDNAを準備する</li></ol> DNA超音波処理 DNAの超音波処理とMeDIPのプロトコルは、管壁へのタンパク質の非特異的結合を防ぐためにsiliconziedチューブで実行する必要があります。 DNAサンプル用に最適な超音波処理時間は、ゲル電気泳動（ステップ27）から決定されたDNAサンプルの断片化の程度に基づいています。例えば、培養細胞から抽出したDNAは非常に高分子量でなければならない、と続いて、しばしば部分的に分解されるアーカイブのサンプルから抽出したDNAよりも多くの超音波処理が必要になります。サンプルが均一に高分子量のものであるなら、それは個別に各サンプルを確認することなく、このプロトコルで説明されているように超音波処理を続行するには、この時点で合理的です。サンプルはアーカイブのサンプルと同様に断片化されている場合、それは300 - 100ベーシスポイントのフラグメントを得るために超音波処理時間を減少させることによって可能性が高い超音波処理の手順を適応させるために必要となります。あなたが部分的に分解されているサンプルを処理することが予想される場合は、超音波処理パラメータの最適化は重要なものから代表的なサンプルで実行することができます。ゲル電気泳動（ステップ27）から観測されたDNA断片化の程度に基づいて、実験者はサンプルのために最適な超音波処理の時間を事前に決定することができます。ここでは、高分子量のDNAを用いて自動化された超音波処理装置（DiagenodeのBioruptor、UCD - 200 TM）と超音波処理によって約300〜1,000 bpのDNA断片を取得する方法を説明します。 <ol><li> Biorupterの水は4でなければなりません℃に</li><li>自動設定で7分（30秒、最大パワーで30秒オン、オフ）のために超音波処理。 </li><li>免疫沈降（IP）反応のためにシリコーン1.7ミリリットルの遠心管に超音波処理した製品や場所、800 ngの（40μl）を取り外します。 </li><li>入力として機能するように、残りの200 ngの（10μl）を（IN）（4℃で保存）リファレンスDNAを脇に置きます。 </LI&gt; </ol>メチル化DNAのIMMUNOPRECIPITATON <ol><li>水浴中で10分間95℃でIP反応（800 ng）のために使用されるDNAを変性させる。 </li><li>氷上で直ちに冷却してください。次のステップに進む前に（氷の上で約5分）完全にDNAを冷ます。 </li><li> 5μgのモノクローナル抗体を追加。 </li><li> 500μlの最終容量にIPバッファを（このプロトコルを通じて、室温で使用される）を追加します。 </li><li>チューブホルダーを回転で4で2時間℃でインキュベートする。 </li><li>手順5が完了する直前に、（ステップ6-11）洗浄してダイナビーズを準備。最初に、ボルテックスでバイアルに徹底的にビーズを懸濁します。 </li><li>新しいシリコーンチューブ（例えば、8の反応を行う場合、9の十分なビーズを除去する、すなわち、270μL）に、反応プラス1あたりの再懸濁したダイナビーズ30μlを（〜2 × 10 7）転送する。 </li><li>室温で2分のための磁気ラック上にチューブを置きます。 </li><li>上清をピペットで。上清を除去する際、ピペットの先端で内部の壁（ビーズが磁石に引き付けている）に対して、ビーズには手を触れないでください。 </li><li>磁石からチューブを外し、IPバッファの過剰量（750μlの- 1000μL）でビーズを再懸濁します。バック室温で2分のための磁気ラック上にチューブを置きます。 </li><li>一回以上の洗浄を繰り返し、次にステップ7で取り外した元のボリュームにIPバッファーで洗浄したビーズを再懸濁します。 </li><li>各IP反応液に洗浄ダイナビーズ30μlを追加する</li><li> 4℃で2時間回転管ホルダー℃でインキュベートする</li><li>インキュベーションが完了した後、室温で2分のための磁気ラック上にチューブを置きます。 </li><li>上清をピペットで。ピペットの先端でチューブの内壁を（ビーズが磁石に引き付け場所）には触れないでください。 IP緩衝液500μlを追加。チューブの内容物を混合し、2分のための磁気ラックに戻す。 500μlのIPバッファーで1回洗浄繰り返します。 </li><li>最後の洗浄上清を除去した後、消化バッファ400μlでビーズを再懸濁します。 </li><li>プロテイナーゼK100μgのとの反応を治療し、50℃で一晩インキュベート℃、 </li></ol>免疫沈降したDNAの精製<ol><li> 500μlの1時01フェノール/クロロホルムpHを7と完全にボルテックスを追加。 </li><li>室温で10分間13,000 gでスピン。 </li><li>新しいチューブに水（上部）分数を取り外します。 </li><li>水溶液と有機層との間の相間が曇って表示されている場合は手順1〜3を繰り返します。 </li><li> 1 / 10量の3 M酢酸ナトリウム（40μl）を、ボルテックスを追加。 </li><li> 1グリコーゲンの溶液（20μg/μLの）と渦を追加。 </li><li> 100％エタノール、渦、および20分まで-20℃での場所の2倍量（1000μL）を追加します。 </li><li> 4℃で20分間13,000 gでスピン℃、 </li><li>エタノールを削除する、パルスのスピン、そして残留エタノールを除去します。 </li><li>洗浄に500μlの冷70％エタノールを加えます。 4℃で20分間13,000 gで軽くボルテックスし、そしてスピン℃に</li><li> 70％エタノール、パルスのスピンを削除し、ピペッティングにより残留エタノールを除去します。 </li><li>空気が残留エタノールの痕跡をすべて削除するには、室温で10分のためのオープンキャップでペレットを乾燥させます。 </li><li>のdH 2 0滅菌10μlに再懸DNAペレット。 </li></ol> PCRによる検証<ol><li>あなたのMeDIPの手順は通常のヒトDNAを（男性または女性）を使用してMeDIPのプロトコルを実行することにより、ワーキング、その後、メチル化に富むことが知られている地域をアッセイしていることを確認するためにテストすることができます。 </li><li> PCRチューブにMeDIPの製品の30％、および他のPCRチューブにMeDIP製品の別の30％を削除します。 </li><li> PCRチューブにDNAの10ngの、および他のPCRチューブにDNA中の10 ngを置く。これで、4つの別々のPCR反応をセットアップする必要があります。 </li><li> PCRは、H19とCTRLプライマーを用いて行うには、表1に示した。 </li><li>表2に示すように12.5μL、総体積とのPCR反応の2つのmastermixes（各プライマーセットで1つずつ）を用意。 </li><li>表3に記載された条件を用いてPCRをThermocycle。 </li><li>可視化のための2％アガロースゲルでPCR産物の5μlを実行します。 </li><li>成功MeDIPの予想結果を表4に示す。 </li></ol>テーブル表1：H19とPCRの検証のためにCTRLプライマー。 <table border="1"><tbody><tr><td>プライマーセット</td><td>フォワードプライマー</td><td>リバースプライマー</td><td>予想される製品のサイズ</td></tr><tr><td> H19 </td><td> H19_F 5&#39; - cgagtgtgcgtgagtgtgag </td><td> H19_R 5&#39; - ggcgtaatggaatgcttgaa </td><td> 174 bpの</td></tr><tr><td> CTRL（コントロール） </td><td> CTRL_F5&#39; - gagagcattagggcagacaaa </td><td> CTRL_R 5&#39; - gttcctcagacagccacattt </td><td> 139 bpの</td></tr></tbody></table>表2。 PCR反応のMastermixes。 <table border="1"><tbody><tr><td></td><td> H19ミックス（あたりRXN） </td><td> CTRLミックス（あたりRXN） </td></tr><tr><tD&gt;のddH 2 O </td><td> 6.875 </td><td> 6.875 </td></tr><tr><td> 10Xバッファー</td><td> 1.25 </td><td> 1.25 </td></tr><tr><td> dNTPミックス（10 mMの各） </td><td> 0.25 </td><td> 0.25 </td></tr><tr><td>のMgCl 2（50mMの） </td><td> 0.25 </td><td> 0.25 </td></tr><tr><td>プライマー（H19_F / RまたはCTRL_F / R）（10μMの各F / R） </td><td> 0.625 </td><td> 0.625 </td></tr><tr><td>プラチナTaqポリメラーゼ</td><td> 0.25 </td><td> 0.25 </td></tr></tbody></table>表3。 PCR thermocyling条件。 <table border="1"><tbody><tr><td></td><td> 95 ° C </td><td> 5時00 </td></tr><tr><td rowspan="3"> 40X </td><td> 95 ° C </td><td> 0時30分</td></tr><tr><td> 56℃ </td><td> 0時30分</td></tr><tr><td> 72 ° C </td><td> 0時15分</td></tr></tbody></table>表4。成功MeDIPのPCRの結果を期待。 <table border="1"><tbody><tr><td></td><td colspan="2" align="center">テンプレートDNA </td></tr><tr><td>使用するプライマー</td><td> IN </td><td> IP </td></tr><tr><td> H19 </td><td>正</td><td>正</td></tr><tr><td> CTRL </td><td>正</td><td>マイナス</td></tr></tbody></table>

<ol>
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疾患において重要な役割のDNAメチル化の戯曲の認識が広がりつつあります、この修正を測定するアッセイのため、開発は13、12、3重要性を増してきています。 MeDIP技術は、全ゲノムと遺伝子座特異的レベル6、7の両方でのスクリーニングのための従順なツールです。この手法は、DNAを始めるの限られた量を使用してDNAのメチル化レベルの急速なビューを提供し、異なるソース?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Biorupter sonicator</td>
<td>Tool</td>
<td>Diagenode</td>
<td>UCD-200 TM</td>
<td>&nbsp;</td>
<tr>
<td>1.7ml SafeSeal Microcentrifuge Tubes</td>
<td>Other</td>
<td>Sorenson BioScience</td>
<td>11510</td>
<td>&nbsp;</td>
<tr>
<td>ND 3300 Spectrophotometer</td>
<td>Tool</td>
<td>NanoDrop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibody: Anti-5'-methylcytosine mouse mAb</td>
<td>Reagent</td>
<td>CalBiochem</td>
<td>162 33 D3</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>112-01D</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Tube Rack</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>CS15000</td>
<td>&nbsp;</td>
<tr>
<td>Mini LabRoller</td>
<td>Tool</td>
<td>Labnet International</td>
<td>H5500</td>
<td>&nbsp;</td>
<tr>
<td>IP Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature</td>
</tr>
<tr>
<td>Digestion Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl</td>
</tr>		</tbody>
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		</div>

methylated dna immunoprecipitation

DNAのメチル化パターンの識別には、メチル化が遺伝子発現に大きな影響を持つことが知られているように、エピジェネティクスの研究では一般的な手順であり、通常の開発だけでなく、病気に関与している<sup&gt; 1月4日</sup&gt;。従って、メチル化DNAと非メチル化DNAとを区別する能力は、そのような研究のためにメチル化プロファイルを生成するために不可欠です。メチル化DNA免疫沈降（MeDIPは）興味のサンプルからのメチル化DNAの抽出のための効率的な手法です。<sup&gt; 5月7日</sup&gt;。 DNAのようにわずか200 ngのサンプルは、抗体、または免疫沈降（IP）、反応に十分です。 DNAは、約300〜1,000 bpからサイズの範囲のフラグメントに超音波処理され、そして免疫沈降（IP）と入力（IN）部分に分かれています。 IPのDNAは、その後、熱変性し、モノクローナル抗体は、メチル化DNAを結合させること、抗5&#39;mCとインキュベートされる。この後、一次抗体に対する親和性を有する二次抗体を含む磁性ビーズを添加し、インキュベートする。これらのビーズにリンクされた抗体は、最初のステップで使用されるモノクローナル抗体をバインドします。抗体複合体（メチル化DNA）に結合したDNA溶液から複合体を引き出すために磁石を使用してDNAの残りの部分から分離されている。 IPバッファを使用して数回の洗浄が結合していない、非メチル化DNAを除去するために実行されます。メチル化DNA /抗体複合体は、唯一のメチル化DNAをそのまま残し抗体を消化するためにプロテイナーゼKで消化されています。蛋白質の物質を除去するためにクロロホルム抽出し、後で使用するために沈殿し、水に再懸濁さ：濃縮されたDNAは、フェノールによって精製する。 PCR技術は、IPの増幅産物を分析することにより、地域の欠如に知られているとメチル化配列を含むように知られているため、DNAのMeDIPの手順の効率性を検証するために使用することができます。精製したメチル化DNAは、遺伝子座特異的（PCR）またはゲノムワイド（マイクロアレイやシーケンシング）メチル化の研究に使用し、そのような遺伝子発現プロファイリングとアレイ比較ゲノムハイブリダイゼーションなどの他の研究ツールと組み合わせて適用する場合に特に有用であることができる（ CGH）<sup&gt; 8</sup&gt;。 DNAメチル化へのさらなる調査は、順番に、異常にメチル化DNAを特徴とする、癌などの疾患の新しい治療や予後の研究ツールの開発に便利だと思われる、新たなエピジェネティックなターゲットの発見につながる<sup&gt; 2、4、9-11</sup&gt;。

Watch this Scientific Journal Video about Methylated DNA Immunoprecipitation at JoVE.com

Methylated DNA Immunoprecipitation

このビデオでは、メチル化DNA免疫沈降（MeDIP）のためのプロトコルを示しています。 MeDIPは、選択的に5 - メチルシトシン（抗- 5 MC）のための特異性を有する抗体を用いてゲノムDNAのサンプルからメチル化DNA断片を抽出する二日間の手順です。

メチル化DNA免疫沈降

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Research

JoVE Journal

Biology

23.2K ビュー.  BC Cancer Research Centre. このビデオでは、メチル化DNA免疫沈降（MeDIP）のためのプロトコルを示しています。 MeDIPは、選択的に5 - メチルシトシン（抗- 5 MC）のための特異性を有する抗体を用いてゲノムDNAのサンプルからメチル化DNA断片を抽出する二日間の手順です。

ビデオ: Methylated DNA Immunoprecipitation

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

生物学

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

ルシフェラーゼ免疫系による抗体のプロファイリング（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

プロービングDNA安定同位体（DNA - SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM：フローサイトメトリーにより検出免疫沈降

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

を使用してクロマチン免疫沈降（ChIP）ショウジョウバエ組織

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

SeqA-DNA複合体の結晶化のためのDNA二重鎖の反復最適化

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

バイオマスの制限額を使用して効率的なクロマチン免疫沈降

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ハイスループットシーケンシングのための高品質クロマチン免疫沈降DNAテンプレート（チップ-SEQ）の生成

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...