我々 は特に IGBMC 動物施設のスタッフのスタッフ IGBMC 高スループット シーケンス機能、「フランス Génomique」コンソーシアム (ANR10-INBS-09-08) のメンバー、IGBMC のすべての一般的なサービスをありがちましょう。この作品は、CNRS、INSERM、AFM、リーグ国立 contre le 癌からの補助金によって支えられた、フランスの国営ラベル ANR-10-アイデックス-0002-02、Labex INRT ANR-10-アイデックス-0001-02 Investissements d'Avenir プログラムの下で ANR を通じて基金とANR-AR2GR-16-CE11-009-01。資金提供者には、研究デザイン、データ収集と分析、意思決定を発行し、または原稿の準備の役割はなかった。S.J にミニステール デ期リーグ国立 contre le 癌と ANR-AR2GR-16-CE11-009-01、V.U によって支えられらデ ラ凝った。ID は 'デフランス labellisée' リーグのナシオナル contre le がんです。

マウスは、ケアおよび実験動物の使用に関する制度のガイドラインに従い、国立動物診療ガイドライン (欧州委員会指令 86/609/CEE; に従って保たれました。フランス語令 no.87 848)。フランス国民の倫理委員会で承認されたすべてのプロシージャ。
 1 です。 筋肉組織分離 頚部転位によって <ol><li>犠牲 1 つアダルト 6 に 8 週齢マウス。Sterlise 70% エタノールで洗浄によって肢細かい点はさみと鉗子を使用して後肢の筋肉 (大腿四頭筋腓腹筋、脛骨前方) を解剖と 。
	注: 1 つのマウスの組織の約 500 mg をもたらすべきである</li>。
	<li>1 mL の小型の同種の準備に冷たい低張性バッファーを含む 2 mL 試験管に筋肉をミンチ (&#60; 2-3 mm 3) を使用して個はさみの罰金し、5-10 分のための 4 ° C でベンチ トップ アジテーターに関する振動管を残す</li>
</ol> 2。組織溶解 注: すべてのバッファー組成の 表 1 を参照してください。
<ol><li>14 mL 丸底チューブにホモジュネートを転送し、5 mL の冷たい低張性バッファー (EDTA 無料プロテアーゼ抑制剤のカクテルそして PMSF) で再懸濁します。
	<ol><li>Homogenise 14 mL の丸底チューブに 15-30 秒の緩い dounce (3 分以内 20 30 ストローク) または機械的組織ホモジナイザーを使用してみじん切りにした筋肉組織 。
		注: この段階では光学顕微鏡検査によって評価される換散の効率と追加の中断を行うことが必要な場合です</li>。
	</ol></li> <li>15 mL チューブに磨砕液を転送し、冷たい低張性バッファーを使用して 10 の mL にボリュームを埋めます。次のマウスに進む前に以下のように磨砕液を修正します</li>。
</ol> 3。固定 <ol><li>ホルムアルデヒドを最終濃度が 1% に加え、室温で 10 分間振る</li>。
	<li>固定と室温で 5-10 分間揺れを停止する各管で 0.125 M の最終的な集中にグリシンを追加します</li>。
</ol> 4。核の準備 注: すべてのバッファー組成の 表 1 を参照してください。
<ol><li>Homogenise 固定ライセート使用緩い dounce (5 〜 10 ストローク).
	<ol><li>核および細胞残骸を取得する 15 mL チューブ 1,000 x g で 4 ° C で 5 分間遠心し転送します</li>。
	</ol></li> <li>上澄みを除去し、5 mL の新鮮な低張バッファーでペレットを再懸濁します。70 μ m 携帯こし器を通って 50 mL のチューブに液をフィルターします。再濾液を 40 μ m セル ストレーナー フィルターします</li>。
	<li>は、核のペレットを取得する 4 ° C で 5 分間 15 mL チューブ 1,000 × g で遠心分離し濾液を転送します 。
	注: この段階で核することができますがすぐに超音波処理、またはスナップ乾燥ペレットとして液体窒素で凍結し、-80 に格納されている ° C</li>
</ol> 5。超音波 <ol><li>核ペレット (機械と dounce の両方の均質化のため約 50 μ L) の容積を査定するパックされた核容積の 4 回に 3 まで超音波処理バッファーでそれを再懸濁し、超音波、超音波発生装置を使用しての 4 ° C で 10-15 分の。 
	注: クロマチン (6) 下記のとおりただちに分析または凍結して格納できます-80 で ° C</li>
</ol> 6。次の超音波クロマチンのフラグメント サイズを評価する 注: すべてのバッファー組成の 表 1 を参照してください。
<ol><li>デ-架橋、クロマチンの 30 μ L を 1.5 mL の試験管に取る 5 M 20 μ L を追加し、塩化ナトリウム。500 μ L にボリュームを完了し、65 ° C で一晩インキュベートします。
	<ol><li>次の日、処理を 1 μ L, プロテイナーゼ K を (20 mg/mL 在庫)、42 ° C 実行フェノール/クロロホルム クロロホルム抽出で 1 時間 0.5 M の EDTA の 10 μ L 2 M トリス pH 6.8, および 10 μ とナトリウムの acetat の 1 つのボリュームで DNA を沈殿させるe (3 M) と 1 時間-80 ° c または-20 で一晩 100% エタノールの 2 つの容積 ° C</li>
	</ol></li> <li>13,500 x g で 15 分間、上清をデカントでの遠心分離で DNA をペレットし冷たい 70% エタノールや 5 分上清をデカント、再び遠心分離で徹底的にペレットを洗浄し、ペレットを風乾します</li>。
	<li>は、TE バッファーの元のボリューム (30 μ L) でペレットを再懸濁します。260 nm/280 nm の吸光度によって分光光度計で DNA 濃度を測定します。クロマチンのフラグメントのサイズを評価するために電気泳動を行いピペットの 1.5% の agarose のゲルの別のレーンに DNA のサイズの梯子とともに DNA の 500-1,000 ng 。
	注: フラグメント サイズは 200-500 塩基対の間にする必要があり、対応するまたは悪い-断片化されていない DNA 検出の高分子量フラグメントはないはず。クロマチン ソリューション (手順 5) の長い時間再超音波し、再分析が最適なサイズになるまで上記のようことが必要な場合です</li>。
</ol> 7。クロマチン免疫沈降 (チップ) 注: すべてのバッファー組成の 表 1 を参照してください。
早かった 1 mL のエタノールで 50% スラリーによる <ol><li>蛋白質 G ブロック セファローズ ビーズ。ペレット、ベンチトップで 1 分間 400 × g で遠心分離によるビーズ遠心 400 x g で TE バッファー遠心の 1 mL で洗いし、ワシントンを繰り返す
	<ol><li>酵母 tRNA (在庫 10 mg/mL) の 2 回目の遠心分離、BSA (株式 20 mg/mL) の 25 μ L で希釈バッファーを内蔵の 1 mL に再停止と 20 μ L 後。4 回転 (40-60 rpm) によって、少なくとも 2 h 使用する前にビーズをブロック ° C</li>
		<li>クロマチン (手順 5) の希釈 50 μ g 8 〜 10 回チップ希釈バッファーを使用しています。ブロック ビーズ スラリーの 50 μ L を追加し、4 回転 (40-60 rpm) 2 時間インキュベート ° C. 遠心済みクリア クロマチンを取得して新鮮なチューブに移転 4 ° C で 400 x g で</li>。
	</ol></li> <li>免疫沈降、一次抗体 (例えば クロマチンの 10 μ g あたり 1-2 μ g) の適切な量を追加し、4 回転で一晩インキュベート ° C 
	注: 最適な抗体の量は製造業者によって推薦されるかもしれないまたは最適な濃縮が観察されるまでさまざまな量の抗体を持つチップ qPCR を実行することによって、経験的に決定できます。蛋白質 G セファローズ可能性があります使用される抗体のサブタイプによって蛋白質 A セファローズに置き換えることも</li>。
	<li>次の日ブロック ビーズを追加し、4 回転 (40-60 rpm) で 1 時間インキュベート ° C. 遠心ビーズをペレット、上清を除去し、チップを開始する 20-30 s の 400 x g で洗うです</li>。
	<li>チップの洗浄: バッファーが次の洗浄を実行: 低塩バッファーに一度し LiCl バッファーで 2 回、高の塩バッファーで 2 回、そして TE バッファーで 2 回。
	<ol><li>4 ° c (40-60 rpm) 回転で 10 分間各洗浄を行い、ベンチトップ遠心分離機で 20-30 s の 400 × g で遠心分離によって洗うたび間ビーズをペレットします</li>。
	</ol></li> <li>溶出、削除最後の洗浄と揺れながら室温で 15 分間 (新鮮な) 250 μ L 溶出バッファーのビードを再懸濁します</li>。
	<li>遠心分離機 x g と収集新鮮なチューブで溶出液 400 で。この 2 回のステップを実行して実行・ デ ・架橋溶出液 20 μ L NaCl 5 M と 1 μ L RNase A で一晩 (10 mg/mL)、扱うプロティナーゼ K のあり、フェノール/クロロホルム クロロホルム バキューム手順 6 のように行動します</li>。
	<li>TE バッファーの 50 μ L で再懸濁します、チップの品質をチェックする標準プロトコル 15 に従ってチップ qPCR の因数を使用します</li>。
</ol>

<ol>
	<li>Johnson, D. S., Mortazavi, A., Myers, R. M., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+in+vivo+protein-DNA+interactions.">Genome-wide mapping of in vivo protein-DNA interactions.</a> Science. 316 (5830), 1497-1502 (2007).</li><li>Furey, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+and+beyond:+new+and+improved+methodologies+to+detect+and+characterize+protein-DNA+interactions.">ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions.</a> Nat Rev Genet. 13 (12), 840-852 (2012).</li><li>Wade, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+Transcription+Regulatory+Networks+with+ChIP-seq+and+RNA-seq.">Mapping Transcription Regulatory Networks with ChIP-seq and RNA-seq.</a> Adv Exp Med Biol. 883, 119-134 (2015).</li><li>Alpern, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAF4,+a+subunit+of+transcription+factor+II+D,+directs+promoter+occupancy+of+nuclear+receptor+HNF4A+during+post-natal+hepatocyte+differentiation.">TAF4, a subunit of transcription factor II D, directs promoter occupancy of nuclear receptor HNF4A during post-natal hepatocyte differentiation.</a> Elife. 3, e03613 (2014).</li><li>Martianov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+occupancy+of+an+extended+repertoire+of+CREM+and+CREB+binding+loci+in+male+germ+cells.">Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells.</a> BMC Genomics. 11, 530 (2010).</li><li>Achour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+identity+genes+regulated+by+super-enhancers+are+preferentially+down-regulated+in+the+striatum+of+Huntington's+disease+mice.">Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice.</a> Hum Mol Genet. 24 (12), 3481-3496 (2015).</li><li>Joshi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TEAD+transcription+factors+are+required+for+normal+primary+myoblast+differentiation+in+vitro+and+muscle+regeneration+in+vivo.">TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo.</a> PLoS Genet. 13 (2), e1006600 (2017).</li><li>Ohkawa, Y., Mallappa, C., Dacwag-Vallaster, C. 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著者は、彼らは競合する金銭的な利益があることを宣言します。

ここでアダルト マウス骨格筋このクロマチンは筋線維核内転写因子結合と共有結合のヒストンの修正を検出チップ実験に適しているショーからクロマチンを準備するための新しいプロトコルについて述べる。このプロトコルには、いくつかの重要な手順が含まれます。最初の dounce または機械的剪断によって実行することができます組織混乱です。機械的剪断はより速くより再現性の高い、?...

クロマチン免疫沈降 (チップ) の量的なポリメラーゼの連鎖反応 (qPCR) に結合し、転写を研究する選択の方法と様々 な組織における遺伝子発現のエピジェネティック制御高スループット シーケンス (チップ seq) となっています。細胞タイプ1。この技法は、ゲノムは共有結合性クロマチン修飾ヒストン バリアントの占有率、転写因子結合2,3プロファイリングできます。
培養細胞からチップを実行することが確立している哺乳類動物組織からチップのままより困難です。チップのクロマチンを準備、組織切片・細胞の種類ごとに最適化が必要ないくつかの重要な手順が含まれます。クロマチンは、培養細胞の細胞の溶解液や、原子核の次の浄化から用意できます。哺乳類ティッシュの場合効率的な換散、ホルムアルデヒド固定および核の精製は、クロマチンの最適な回復を確保するために重要です。また、その浄化の前後に核を修正するかどうかの選択は実験的に決定します。これらの障害にもかかわらず、肝臓、精巣、または脳4,5,6などの組織からチップに正常に行われています。骨格筋の場合このような物理的に抵抗力がある組織、構造タンパク質の含有量が高いと核の分離を表わすは特に困難です。この特異性を考えると、他の組織のために最適化されたプロトコルを与えない満足のいく結果骨格筋の。
ここで物理的に破壊組織、ホルムアルデヒド固定し核と超音波処理の分離を含むマウス骨格筋組織からチップ グレード クロマチンを分離するプロトコルについて述べる。様々 な転写因子や RNA ポリメラーゼ II、コバレントマテリアルのヒストンの修正7の qPCR チップとチップ seq を実行することによってこの組織からチップに適したクロマチンを準備するこの手法の有効性を示した.
この新しい技術が以前に報告されたプロトコル8時間、転写因子のゲノムのローカライゼーションの変更中に長いコラゲナーゼ消化手順を構成するよりもはるかに高速と遺伝子発現の変化が起こるかもしれない。速さを核隔離され、固定は、ここで説明する方法をより忠実にネイティブのゲノムの占有状態をキャプチャするクロマチンを準備する特に魅力的になります。また、筋肉組織からクロマチンの分離または蛋白質の抽出のための他の方法が記述されている8,9,10され選択した遺伝子、チップ seq チップ qPCR 実験用データは、それらを使用して報告されています。ここで報告メソッドは転写因子やヒストン変更チップ seq に適した、それゆえ必要があります 3/4 C や HiC クロマチン構造キャプチャ ・ アプリケーションに適しても。

<table>
	<tbody>
		<tr>
			<td>T 25 digital ULTRA-TURRAX</td>
			<td>T 18 ULTRA-TURRAX</td>
			<td>0009022800</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>SIGMA-ALDRICH CHIMIE SARL</td>
			<td>P7626-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail-EDTA Free</td>
			<td>Roche Diagnostics</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Sepharose beads</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P-3296</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P-2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers 70um</td>
			<td>Corning BV</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers 40um</td>
			<td>Corning BV</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde EM grade</td>
			<td>Euromedex</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Fischer Scientific</td>
			<td>12091039</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P3803</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>B4287-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast tRNA</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>R5636</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Blue</td>
			<td>AMIBION</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Pol II ChIP Antibody</td>
			<td>Santa Cruz</td>
			<td>SC-9001</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27ac ChIP Antibody</td>
			<td>Active Motif</td>
			<td>39133</td>
			<td></td>
		</tr>
		<tr>
			<td>Tead4 ChIP Antibody</td>
			<td>Aviva Systems Biology</td>
			<td>(ARP38276_P050)</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>E220 Focused Ultrasonicator</td>
			<td>Covaris E220</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additional items</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Loose Dounce</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml and 50 ml falcon tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml round bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml and 2 ml Eppendorf tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m and 40 &#956;m cell strainers (Corning)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

improved protocol for chromatin immunoprecipitation from mouse skeletal muscle

成体マウス骨格筋構造蛋白質の含有量が高い物理的に抵抗性組織からクロマチンの準備のための効率的かつ再現性のあるプロトコルについて述べる。大人のマウスから切り裂かれた四肢の筋肉物理的、機械的均質化またはミンチと細胞ライセートのホルムアルデヒド固定前に低張性バッファー内の douncing の組み合わせによって中断されます。固定の核は機械的均質化または douncing 細胞の残骸を削除する連続ろ過のさらなるサイクルによって精製しました。精製された核は、すぐに、後の段階で凍結後する超音波に処理できます。クロマチンが効率的に超音波処理および転写因子、RNA ポリメラーゼ II、および共有結合のヒストンの修正のプロファイルによって示されているように、クロマチン免疫沈降実験に適した取得します。このプロトコルにより作製したクロマチンを使用して検出されたバインディング イベントは、主に筋線維核クロマチン繊維関連の他の衛星と血管内皮細胞からの存在にもかかわらずで行われているものです。このプロトコルはそのため成体マウスの骨格筋における遺伝子発現制御の研究に適応です。

核を分離するには、18,000 と 22,000 の rpm (材料の表を参照) で、15 または 45 s のため解剖とみじん切りにした筋肉組織の力学的均質化を行った。すべての条件で核組織破片から分けることができるが、収率は低い速度 (図 1 aB) を使用して最適です。核は、3-5 分 (図 1 aと示されていないデータ) の dounci...

Watch this Scientific Journal Video about Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle at JoVE.com

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

クロマチン免疫沈降マウス骨格筋からの改善されたプロトコル

クロマチン免疫沈降による筋線維の遺伝子の規則の研究に適応した成体マウス骨格筋からクロマチンの準備のための新しいプロトコルが表示されます。

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

improved-protocol-for-chromatin-immunoprecipitation-from-mouse

Research

JoVE Journal

Biochemistry

8.4K ビュー.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. クロマチン免疫沈降による筋線維の遺伝子の規則の研究に適応した成体マウス骨格筋からクロマチンの準備のための新しいプロトコルが表示されます。

ビデオ: Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

ヒト僧帽弁からのタンパク質抽出のための最適化プロトコル

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

生化学

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

クロマチン免疫沈降のための架橋された骨格筋からの高品質核の調製のためのろ過に基づく方法

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

西部のしみが付くプロトコル造血幹細胞の数が少ない

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

標的タンパク質の翻訳後修飾の内因性プロファイルを識別するために包括的な免疫沈降濃縮システムを活用

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

測定と酸素消費量全体の解釈飛ぶ頭セグメント

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

多重共免疫沈殿を用いたタンパク質相互作用ネットワークダイナミクスの定量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

大規模核インターロームプロファイリングのためのラベルフリー免疫沈降質量分析ワークフロー

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

スイマーの心呼吸評価のための急速に増加するテザド水泳最大プロトコル

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

細菌におけるRIBO-seq:NGSシーケンシングのためのサンプル収集とライブラリ調製プロトコル

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

骨格筋ミトコンドリアのタンパク質輸入能の測定

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...