Name: chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae
Uploaded: 2017-12-29T15:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

著者は、役に立つ議論のため緑のラボのメンバーに感謝したいと思います。この作品は、NIH の補助金 R03AG052018 と E.M.G. に R01GM124342 によって部分で支えられました

1. 事前バインド磁性ビーズに抗体
<ol>
	<li>タンパク質 A/G 磁気ビーズを混ぜるし、磁気ビーズ 1 つ免疫沈降 (IP) サンプルごとの 20 μ L を 1.5 mL チューブに転送します。 注: ときにビーズをピペッティング ワイド口径、低維持のヒントを使用します。</li>
	<li>マグネット スタンドにビーズとチューブを置き、側管の収集にビーズを許可します。上澄みを除去します。</li>
	<li>冷たいトリス緩?...

<ol>
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ここで説明したプロシージャ免疫沈降による酵母細胞の変更されたヒストンに関連付けられている DNA の効率的な復旧できます。これはローカル濃縮を決定する対象領域または特定のヒストンの修正の枯渇を増幅するプライマーを使用して qPCR が続きます。ほぼ 20 年前メソッドとして開発されているにもかかわらずチップのまま異なるゲノム領域で、多様な条件下でのヒストン変更状態を調?...

ヒストンの動的の翻訳後修飾 (PTM) は、転写、複製、DNA 修復1,2を含む多くの dna のプロセスに対して重要な規制メカニズムです。豊かさと変更されたヒストン併用、これらのプロセスの正確な局在を決定する機能がセルに異なる条件の下で、規制を理解したがって欠かせません。主茎では、DNA 蛋白質の相互作用の生化学的研究から特に体外法化学塗料の硬化剤、方法としてクロマチン免疫沈降 (チップ) の開発を評価する必要性と相まって、蛋白質 DNA 相互作用の体内とゲノム3,4、5の特定の領域での動的な性質。(QPCR) の量的な PCR とシーケンス テクノロジーの進歩もチップ実験で DNA 蛋白質の相互作用を解剖するための強力なツールとなって全体のゲノムの定量的な比較を実行する能力を拡大しています。複数のレベル。
現在、チップはゲノムのクロマチン パミンに興味を持って、研究グループの必要なメソッドとして変更されたヒストンおよび特定の genomic 位置の間で物理リンクに直接問い合わせることに匹敵するメソッドがないです。vivo。さまざまな科学的な質問の規模、コスト、これらのアプローチがアドレスがありますゲノム6,7中のヒストンの修正をマップする次世代シーケンシングを使用してこのメソッドのバリエーションはありますが、テクニカル リソースは、いくつかの研究グループの制限があります。さらに、対象となるチップ qPCR はこれらを補完するために必要な方法で両方を提供することによってアプローチを最適化チップ プロトコル シーケンスの前に、エピゲノムのデータセットの結果を検証します。質量分析法に基づくアプローチ8,9,10,11, をただし、浮上してもゲノム領域に関連付けられているマークがあるヒストンの完全な補完を識別するこれらアプローチに関するゲノムの領域を検出できる、技術的な専門知識と研究のすべてのグループに使用できなくなります計装を必要といくつかの制限があります。したがって、チップは豊かさとエピジェネティクス、クロマチンとゲノム機能の制御に興味があるすべての研究グループの多様な条件下でのヒストンの修正の分布を分析するための基本メソッドです。
ここでは、出芽酵母を用いたチップ モデル酵母 (出芽酵母)クロマチンでヒストン Ptm の分布を調査する手法について述べる。このアプローチは、酵母を開発し、多様なモデル システム12,13にも適用チップ プロトコルのコア コンポーネントの数に依存します。変更されたヒストンと DNA、細胞間の相互作用は、ホルムアルデヒドで架橋によって保持されます。次の試薬、クロマチン フラグメントによって可溶性に均一サイズの断片にミクロコッカスヌクレアーゼで消化。商業やラボで生成された抗体、変更されたヒストンの免疫沈降を実行され、任意の関連付けられた DNA を分離して、qPCR (図 1) を使用して特定のゲノム領域で濃縮を分析します。多くのヒストンの修正、この議定書から得られた DNA の量は qPCR で 25 以上の異なるゲノム遺伝子をテストするため十分です。
このチップ メソッドは、汎用性の高い複数の変異株や環境条件は、1 つのヒストン修飾の分布を監視または野生型細胞ゲノムの遺伝子の数で複数のヒストンの修正をテストするためです。さらに、プロトコルの多数のコンポーネントは、いずれか高いまたは卑しい-豊富なヒストンの検知を最適化するために容易に調節可能。最後に、出芽酵母で変更されたヒストンのチップを実行する他のシステムで主に使用される抗体の特異性の主要なコントロールを使用する機会を提供します。すなわち、酵母に変更、対象とヒストンの残基の点突然変異を運ぶ生成され、いくつかのケースでは、のみ特定のヒストン残渣の変更を促進する単一酵素 (e.gヒストンのリジン。メチル化酵素)。したがって、チップは、いずれか、ヒストン変異体や酵素削除系統程度抗体の非特異的結合が発生して偽陽性の結果を生成する可能性がありますを試金する実行できます。このコントロールは、新しく開発された抗体、特に貴重なも他のシステムで、使用前に保存されたヒストン修飾の抗体の特異性を検証に使用することがあります。このアプローチは、(モノ-、ジ -、tri-メチル化) などの別の修正状態の間で区別する抗体の特異性をテストするのには他の方法を補完する変更されたペプチドの配列をプローブとヒストンの西部のしみの実行を含むまたは定義された変更とヌクレオソーム。全体的にみて、出芽酵母におけるチップはヒストン Ptm ゲノム全体のダイナミクスを評価して、その調節を支配するメカニズムを解剖する有力な手法であります。

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast Extract</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">Y20025-1000.0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Peptone</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">P20250-1000.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Dextrose</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP350-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycine</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>AC12007-0050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Tris</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">0497-5KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">NaCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP358-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">74385</td>
			<td>Equivalent to NP-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">MgCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">CaCl2</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">20899-25G-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">LiCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">AC413271000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Dodecyl Sulfate</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">M107-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Deoxycholate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">30970-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Acetate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">S2889</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">NaHCO3</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast protease inhibitor cocktail</td>
			<td>VWR</td>
			<td>10190-076</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td>
			<td style="width:301px;">VWR Life Science</td>
			<td>97064-824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ethanol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Nuclease-Free Water</td>
			<td style="width:301px;">VWR</td>
			<td style="width:164px;">100720-992</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micrococcal Nuclease</td>
			<td>Worthington Biochemical</td>
			<td>LS004797</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycogen</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">R0561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Proteinase K</td>
			<td>Research Products International (RPI)</td>
			<td>P50220-0.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">RNase A&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R6513-50MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Bradford Assay Reagent</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">23238</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;BSA Standard 2 mg/mL</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>23210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4</td>
			<td>EMD Millipore</td>
			<td>04-858</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4K16ac</td>
			<td>EMD Millipore</td>
			<td>ABE532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3</td>
			<td style="width:301px;">Abcam</td>
			<td style="width:164px;">ab1791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3K4me2</td>
			<td>Active Motif</td>
			<td>39142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;High Rox qPCR Mix</td>
			<td>Accuris qMax Green, Low Rox qPCR Mix</td>
			<td>ACC-PR2000-L-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Protein A/G Magnetic Beads</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>88803</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">magnetic stand for 1.5mL tubes</td>
			<td>Fisher Scientific</td>
			<td>PI-21359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acid-Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td style="width:164px;">G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Microtube Homogenizer</td>
			<td style="width:301px;">Benchmark</td>
			<td>D1030</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">2.0 mL screw-cap tubes with sealing rings</td>
			<td>Sigma-Aldrich</td>
			<td>Z763837-1000EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Gel loading tips</td>
			<td>Fisher Scientific</td>
			<td>07-200-288</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cuvettes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">50-476-476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Parafilm</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">50 mL conical tubes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">384-Well PCR Plate</td>
			<td>Fisher Scientific</td>
			<td>AB-1384W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Gyratory Floor Shaker</td>
			<td style="width:301px;">New Brunswick Scientific</td>
			<td style="width:164px;">Model G10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Spectrophotometer</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">ND-2000c</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">&#160;Real-Time PCR Detection System</td>
			<td style="width:301px;">Bio-Rad</td>
			<td style="width:164px;">1855485</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae

アセチル化、メチル化、リン酸化などの翻訳後修飾ヒストン修飾 (Ptm) は、一連のセルによって受信した信号への応答でこれらのマークの削除を追加または酵素によって動的に調整されます。これらの PTM が遺伝子発現制御などのプロセスの規則への主貢献者および DNA 修復します。クロマチン免疫沈降 (チップ) は、豊かさとセルに多様な摂動に対してゲノム全体にわたって多くのヒストン Ptm のローカリゼーションを解剖するため手段アプローチをされています。ここでは、出芽酵母酵母 (出芽酵母)からファーストクリーニング変更されたヒストンのチップを実行するための汎用性の高いメソッドを説明します。このメソッドはホルムアルデヒド治療イースト文化の DNA やタンパク質の架橋結合に依存しているミクロコッカスヌクレアーゼ、によってクロマチン フラグメントの可溶化とヒストン DNA の免疫沈降を破って、ビーズで酵母溶解液の生成複合体。興味のヒストン マークに関連付けられている DNA は、精製は、ゲノム全体の複数の座位で濃縮を評価する定量的 PCR 分析を受けます。野生型と変異酵母ヒストン マーク H3K4me2 と H4K16ac の局在化を探る代表的な実験、データ分析と解釈を示す説明します。このメソッドは、さまざまなヒストン Ptm に適していて、異なる突然変異系統またはそれに異なる条件下でのクロマチン動態の変化を調査するための優れたツールを作り、多様な環境ストレスの存在下で実行できます。

このプロトコルの 1 つの主要コンポーネントは、ステップ 10 で説明したよう、可溶性フラグメントにクロマチンを消化するために使用ミクロコッカスヌクレアーゼ (MNase) の濃度が最適化されます。これは興味のゲノム領域でのヒストンの修正の分布に関する高解像度データを取得するため重要です。MNase 滴定は、主にモノラル-ヌクレオソーム クロマチンの可溶性画?...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

酵母からヒストン修飾のクロマチン免疫沈降 (チップ)

ここでは、出芽酵母酵母から変更されたヒストンのクロマチン免疫沈降のためのプロトコルについて述べる。沈澱 DNA はその後量的な PCR 豊かさとゲノム全体ヒストン翻訳後修飾のローカリゼーションを調査する使用されます。

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

The overall goal of this chromatin immunoprecipation protocol is to determine the abundance and localization of histone modifications at defined regions throughout the budding yeast genome. This method can help answer key questions in chromatin regulation, such as how histone modifications are linked to gene expression control and how these modifications change in response to different environmental conditions. An extremely versatile protocol that can be used to investigate many different histone modifications and multiple E strains at one time.Though this method can provide insight into histone modifications, it can also be applied to investigating other chromatin bonding proteins through the use of epitope tags or available antibodies. Start this procedure by growing the yeast cells inoculate 10 milliliters of YPD medium with a single colony of each appropriate strain, and grow it 30 degrees celsius in a shaking incubator at 220 rpm overnight. On the following day after measuring the optical density at 600 nanometers or OD600 of the culture, dilute the culture to an OD600 of 0.2 in 100 mL of YPD in a new flask.Grow the cells at 30 degree celsius in a shaking incubator at 220 rpm until they reach mid log phase. When the cultures have reached mid log phase, add 2.7 milliliters of 37%formaldehyde directly to the medium in each flask for a final concentration of 1%formaldehyde. Transfer the flasks to a shaker at room temperature, and shake at 50 rpm for 15 minutes.Add five milliliters of 2.5 molar glycine to the medium and continue shaking at 50 rpm at room temperature for five minutes to quench the formaldehyde. Subsequently, spin down the cells and wash them as described in the text protocol. Remove the supernatant after the last spin and proceed to making yeast lysates.Re-suspend the cells in one milliliter of cold chromatin amino precipitation or ChIP Lysis buffer with 1 millimolar PMSF and 1:1000 dilution of a yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 milliliter screw cap tube containing 200 microliters of glass beads. Transfer the cells to a bead-beating apparatus for a high speed agitation at four degree celsius.Bead-beat for 30 seconds and ice for one minute. Bead-beat a total of six times, keeping the cells on ice for at least one minute between each 30 second beat-beating. Using a gel loading tip, transfer the lysate to a 1.5 milliliter tube.Centrifuge the lysate at 15, 500 g at four degree celsius for 20 minutes. Discard the supernatant and re-suspend the pellet in 250 microliters of micrococcal nuclease digestion buffer by gently pipetting up and down. Add 2.5 microliters of micrococcal nuclease to each reaction.Mix gently by inverting four to six times and immediately incubate in a 37 degree celsius water bath for 20 minutes. After 20 minutes, stop the reaction by placing the tubes on ice, adding five milliliters of 0.5 molar EDTA for a final concentration of 10 millimolar EDTA and mixing gently by inversion. Centrifuge at 15, 500 g at four degree celsius for 15 minutes.Then, transfer each supernatant to a new 1.5 milliliter tube. For the most consistent results, it is critical to ensure that the same concentration of protein is used in each amino precipitation. Depending on the number of amino precipitation or IP reactions, make a master mix of each micrococcal nuclease released chromatin fraction and ChIP lysis buffer and allocate one milliliter containing 50 micrograms of total protein to individual tubes.Remove 100 microliters from each IP mix to process as the input sample. Freeze the input samples at 20 degree celsius for later use. It is also important to make sure the same amount of beads bound to the antibody is used for each immunoprecipitation.Using a wide board pipette tip, add 20 microliters of magnetic protein AG beads, pre-bound with antibody to each IP sample. Rotate the samples at eight rpm at four degree celsius for three hours. When the IP is done, place the tubes in a magnetic stand and let beads collect on the side of the tube.Use a pipette to remove the supernatant. Add one milliliter of ChIP lysis buffer to the beads and rotate at eight rpm at four degree celsius for five minutes. Then, place the tubes on the magnetic stand and remove the supernatant.In this manner, wash the beads successively with the following buffers for the number of times indicated. ChIP Lysis Buffer, High Salt Wash Buffer, LiCl/detergent Wash Buffer and TE.With the last TE wash, use 0.5 milliliters of TE to transfer most of the beads to a new tube, and then use another 0.5 milliliters of TE to wash the tube and transfer the remaining beads. Add 250 microliters of freshly made ChIP elution buffer to each tube and vortex the solution briefly.Rotate the samples at eight rpm at room temperature for 15 minutes. Place the tubes in the magnetic stand. Carefully transfer the supernatant to a new tube and avoid the beads.Add another 250 microliters of ChIP elution buffer to the beads. Rotate the samples at eight rpm at room temperature for 15 minutes. Carefully and without disturbing the beads, transfer the supernatant to the tube containing the first elution.Thaw the input samples collected earlier and add 400 microliters of ChIP elution buffer to each input sample. To reverse protein DNA cross links, add to both input and IP samples, 20 microliters of five molar sodium chloride, five microliters of glycogen and 12.5 microliters of Proteinase K.Mix well by inverting or flicking the tubes. Incubate the samples in a 65 degree celsius water bath overnight.To begin this procedure, make a QPCR master mix for each set of primers by combining the appropriate volumes of QPCR mix, the forward primer, and the reverse primer. Make DNA master mixes for each DNA sample where one reaction contains 0.5 microliters of DNA and 4.0 microliters of nuclease free water Add 5.5 microliters of the appropriate primer master mix to each well in a 384 well QPCR plate. Spin the plate at 200 g at room temperature for one minute.Then, add 4.5 microliters of the appropriate DNA master mix to each well. Seal the plate, and spin at 200 g at room temperature for one minute. Perform QPCR using a real time QPCR system with the following conditions, 95 degree celsius for three minutes 40 cycles of 95 degree celsius for 10 seconds and 55 degree celsius for 30 seconds and a melting curve of 65 degree celsius to 95 degree celsius with 0.5 degree celsius increments for five seconds.One key component of this protocol is optimizing the concentration of micrococcal nuclease used to digest the chromatin into soluble fragments. This example result from agarose gel electrophoresis of DNA isolated following digestion of the chromatin pellet with varying amounts of micrococcal nuclease shows that 2.5 microliters of enzyme at 20 units per microliter produced predominantly mononucleosomes. This representative ChIP experiment used antibodies against a histone methyl marker H3K4me2 and histone H3 in wild type and set1 knockout cells.Set1 knockout cells lack the methyl transferase that catalyzes the mark The bars indicate the relative enrichment of H3K4me2 at the indicated low sci. The wild type strain showed clear enrichment for H3K4me2 at the five prime end of two genes known to be direct targets of set1, PMA1 and ERG11 whereas no signal was observed in set1 knockout cells. As expected, there was no enrichment of the H3K4me2 mark at TEL07L, the expression of SPR3 has also been reported to be regulated by set1 but these results indicate the regulation is likely to occur by a different mechanism than at PMA1 or ERG11.Once mastered, this technique can be done within four days if it is performed properly. While attempting this procedure, it's important to remember to carefully test the antibody used for specificity with the proper controls. If available, both positive and negative control regions should be probed in the QPCR.Following this procedure, other methods like GP sequencing can also be performed in order to determine the genome by distribution of histone modifications. After watching this video, you should have a good understanding of how to consistently detect the levels of different histone modifications at unique genomic regions with well controlled experiments.

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Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Unit 1

DNA and Chromosome Structure

SCIENTISTS IN ACTION

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

クロマチン免疫沈降法を用いたc-KITリガンドプロモーターの解析

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

遺伝学

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

ChEC-seqとのタンパク質 - DNA相互作用のゲノムワイドマッピング<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

マウスT細胞株におけるクロマチン免疫沈降（ChIP）

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

ネイティブのクロマチン免疫沈降のヌクレオソーム密度解析ライブラリをシーケンスの生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

クロマチン免疫沈降 (チップ) プロトコル低豊富な胚サンプル

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

マウスの褐色脂肪組織のクロマチン免疫沈降

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

哺乳類細胞における微小コッカルヌクレアーゼを用いてクロマチン免疫沈殿アッセイ

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

クロマチン免疫沈降とハイスループットシーケンシング(ChIP-seq)を用いたサルモネラバイオフィルムにおけるCsgD調節標的の発見

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

クロマチン免疫沈降-エキソヌクレアーゼ(ChIP-Exo)を用いたマウス幹細胞由来ニューロンにおけるタンパク質-DNA相互作用の高分解能マッピング

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

マグナペアテオリザエにおけるクロマチン免疫沈降シーケンシング法を用いたヒストン修飾分布のゲノム解析

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...