私たちはKaryn Esser、Koike Nobuya、Park Noheon Parkに助けになることに感謝します。この研究は、NIH / NIGMS（R01GM114424）がS.-HYに、またRobert A. Welch Foundation（AU-1731）とNIH / NIA（R01AG045828）がZC

動物のケアは、施設の動物のケアおよび使用委員会（IACUC）ガイドラインの下で実施され、手順はヒューストンのテキサス大学健康科学センターによって承認された動物のプロトコルに従って行われた。 1.架橋骨格筋からの核の単離<ol><li>前に詳述したように、氷冷リン酸緩衝生理食塩水（PBS）中、約20週齢のC57BL / 6雄マウスから単離した後肢骨格筋を秤量して切り刻む。 注：我々は通常、1つのマウスから1.0〜1.5 gの骨格筋を得る。 <ol><li>氷上で10mLのPBSを含む50mLコニカルチューブに細かい骨格筋組織を置く。 </li></ol></li><li> 4℃で300xgで5分間遠心分離する。 </li><li>慎重に吸引によりPBS上清を除去する。 </li><li> 1、2、3、および4 mLの水を含む参照チューブを使用して、各50 mLチューブのペレット容量を見積もります。 </li><li> 7巻追加PBS中の氷冷した1％ホルムアルデヒドで洗浄し、プローブ組織ホモジナイザー（ 表を参照）を用いて氷上でサンプルをホモジナイズする。 注：オプション：ホモジナイゼーション手順は、低温室で行うことができます。 </li><li>室温で10分間インキュベートすることによって試料を架橋させる。 </li><li> 1Mグリシンを添加して架橋反応を停止させ、0.125Mの最終濃度とし、室温で5分間インキュベートする。 </li><li>サンプルを3,000 xgで5分間4℃で遠心分離する。吸引により上清を除去する。 </li><li>新たに加えたプロテアーゼインヒビター（0.2mMのフェニルメチルスルホニルフルオライド（PMSF））を含有する10mLの氷冷塩基緩衝液（pH7.3,10mM EDTA、10mM KCl、5mM MgCl 2、0.5mM DTTの10mM HEPES-KOH） 、及び10μg/ mLのロイペプチン）を含む。 3,000 gで5分間4℃で遠心分離し、上清を慎重に吸引除去する。 </li><li>ペレットを6mLの溶解緩衝液（10mM HEPES-KOH、p（0.2mMのPMSF、および10μg/ mLのロイペプチン）を含有する10mMのHEPES緩衝液（pH7.5,10mM EDTA、10mM KCl、5mM MgCl 2、0.5mM DTT、1％Triton X- </li><li>試料を予め冷却した15mL Dounceホモジナイザーに移し、氷上で10分間インキュベートする。ゆるやかな乳棒を用いて15回のゆっくりとしたストロークで各試料を氷上でホモジナイズし、密栓した15ストロークで核を放出させる。 </li><li>ホモジネート（6mL）を細胞ストレーナー（孔径：100μm）で濾過し、4mLの溶解緩衝液ですすぐ。 4℃で1,000 xgで10分間遠心します。 5mLの氷冷した塩基緩衝液に再懸濁し、密な乳棒で10回叩いて核を放出させる。 <ol><li>分光光度計（OD260 / OD280）でDNA濃度を測定するために20μLのアリコートを取り出し、必要に応じてトリパンブルーで顕微鏡で観察します（下図参照）（ 図 1A ）。 </li></ol></li><li>サスペンションをフィルタリングする細胞ストレーナ（細孔サイズ：70μm）を用いて、チューブをすすぎ、上記のように2mLの塩基緩衝液で再度濾過する。 </li><li>徐々に細孔サイズ（40μm、30μm、20μmおよび10μm）が減少した細胞ストレーナーで、ステップ1.13と同様にろ過を繰り返します。 注：合計で、6つの孔サイズのストレーナーがステップ1.12-1.14で使用された。 </li><li> 4℃、1,000gで10分間遠心分離する。上清を除去し、ペレットを500μLの塩基緩衝液に再懸濁する。 </li><li>上記のようにDNA濃度を測定する。 注：約200μgの核酸DNAが1匹の動物からの両方の後肢を組み合わせて得られた。必要に応じて、トリパンブルー染色（ストック濃度0.4％、1：5希釈）による顕微鏡観察のために20μLを保存する。核が青く染色される（ 図 1B ）。 </li><li> 4℃、1000gで10分間遠心分離し、上清を捨てる。ペレットを-80℃で保存する。 </li></ol> 2.超音波 &lt;ol&gt; <li>サンプルを500μLのSDS溶解バッファーで解凍し、穏やかなピペッティングで再懸濁します。 </li><li>核のDNA懸濁液を氷上のガラスバイアルに移す。 </li><li>皿状のトランスデューサからの超音波音響エネルギーの集中したバーストで超音波処理を実行します。装置の詳細については、 表を参照してください。次の設定を使用します。強度：5;デューティサイクル：20;温度4〜6℃; 30秒オン/オフ。 注：音波処理の有効性を評価するための例を図2に示します 。 </li><li>超音波処理したクロマチンを1.5mLチューブに移す。 12,000 xgで15分間遠心分離し、上清を新しいチューブに移す。 65℃で一晩逆架橋させるために、超音波処理されたサンプル（〜7-10μg/筋肉サンプル）50μLを新しい1.5mLチューブに移す。残りのサンプルを-80℃で凍結させる。 </li></ol> 3.超音波と定量の評価<ol><li> 1.0を追加保存した50μLの超音波処理されたサンプルに、RNase A（500U / mL RNase A：20,000U / mL RNase T1）を加え、37℃で30分間インキュベートする。 </li><li> 8μLのプロテアーゼK（10mg / mL）を添加し、55℃で30分間インキュベートする。 </li><li> DNA抽出キットを使用してDNAを収穫する。 <ol><li> 5倍量の結合緩衝液を添加し、スピンカラムにかけ、4,000xg、10分間遠心する。 </li><li>パススルーを同じカラムにかけ、再度遠心します。 </li><li>洗浄バッファーで13,000 xg、1分間で2回洗浄する。 </li><li>カラムを乾燥させるために13,000 xgで1分間遠心分離します。 </li><li> 50μLのH 2 Oを加え、13,000×g、1分間遠心分離してDNAを溶出する。 </li><li>パススルーを同じカラムにかけ、再度遠心します。 </li></ol></li><li>分光光度計（OD260 / OD280）でDNAの量を定量する。 0.8％ゲルでサンプルを実行して超音波処理された製品のサイズおよび量（1μg）を評価する（ 図2 ）。 注：平均クロマチン収率は約20％です。 </li></ol> 4.チップ<ol><li>以前保存したクロマチンを-80°Cで解凍し、クロマチンをチューブあたり約100〜120μgに等分します。放射免疫沈降アッセイ（RIPA）緩衝液（20mM Tris-HCl（pH7.4）、150mM NaCl、1mM EDTA、1mM NaF、1mM Na 3 VO 4、1mM PMSF、1×プロテアーゼ阻害剤カクテル（PIC）、0.1％ w / v）、1％トリトン（Triton）X-100）。 <ol><li>各グループに複数のサンプルがある場合は、同量のクロマチンDNAを最終量約100〜120μg/サンプルに合わせます。入力として10％を保存します。 </li></ol></li><li> BSAプレブロック（〜2時間、4℃）プロテインA / Gアガロース（非鶏抗体）またはIgYビーズ（鶏抗体）の40μL（RIPAバッファー中50％v / vスラリー）を添加し、 4℃で3時間回転させた。 </li><li> 1,000 xgで10分間遠心分離し、上清を注意深く新しいチューブに移す。 </li><li>抗体を1μg/25〜100μgのクロマチンDNA。 </li><li>約15 rpmで一晩4℃で静かに回転させます。 </li><li> 10μLのBSAプレブロック（〜2時間、4℃）プロテインA / Gアガロース（非鶏抗体）またはIgYビーズ（鶏抗体）を加え、冷室で2時間回転させます。 </li><li>ビーズを以下のように洗浄する。 RIPA緩衝液1mLで3分間2回洗浄し、1mLの高塩緩衝液（20mM Tris-HCl（pH7.4）、500mM NaCl、2mM EDTA、1mM PMSF、1％Triton X-100）で3分間2回洗浄する。 1mLのLiCl緩衝液（20mM Tris-HCl（pH7.4）、250mM LiCl、2mM EDTA、1mM PMSF、1％Na-デオキシコール酸塩、0.5％NP40）で3分間2回洗浄する。 <ol><li> 1mLのTris-EDTA（TE）緩衝液（10mM Tris-HCl（pH7.4）、1mM EDTA）で3分間1回洗浄する。次いで、溶出緩衝液（20mM Tris-HCl、5mM EDTAおよび0.5％SDS）でDNAを溶出する。 </li></ol></li><li> 1,000gで1分間遠心分離し、上清を慎重に除去する。 </li><li>溶出バッファー50μLでビーズを再懸濁する。 </li><li> 10日間インキュベートする分で65℃で保存した。 </li><li> 12,000 gで5分間遠心分離する。 </li><li>上清を新しいチューブに移し、さらに50μLを加え、12,000gで5分間遠心分離する。最終溶出量は100μLになります。 </li><li>逆架橋およびDNA溶出。 <ol><li>溶出したクロマチンを一晩65℃でインキュベートする。 </li><li> 1.0μLのRNase Aを添加し、37℃で30分間インキュベートする。 </li><li> 8μLのプロテアーゼK（10mg / mL）を添加し、55℃で30分間インキュベートする。 </li><li>製造者のプロトコールに従って、50μLの溶出容量を有するPCRクリーンアップキットを用いてDNAを収穫する。 </li></ol></li><li>先に説明したようにリアルタイムqPCRによるプロファイルを分析する（ 25,26） 。 注：プライマー配列は図3の 凡例にリストされています 。データは、平均±SEMとして示す （ 図3 ）。 </li></ol>

<ol>
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A., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+CLOCK-BMAL1+binding+to+multiple+E-box+motifs+drives+circadian+Dbp+transcription+and+chromatin+transitions.">Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions.</a> Nat Genet. 38 (3), 369-374 (2006).</li><li>Laplante, 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=DEPTOR+cell-autonomously+promotes+adipogenesis,+and+its+expression+is+associated+with+obesity.">DEPTOR cell-autonomously promotes adipogenesis, and its expression is associated with obesity.</a> Cell Metab. 16 (2), 202-212 (2012).</li><li>Blechman, J., Amir-Zilberstein, L., Gutnick, A., Ben-Dor, S., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolic+regulator+PGC-1alpha+directly+controls+the+expression+of+the+hypothalamic+neuropeptide+oxytocin.">The metabolic regulator PGC-1alpha directly controls the expression of the hypothalamic neuropeptide oxytocin.</a> J Neurosci. 31 (42), 14835-14840 (2011).</li></ol>

著者は何も開示することはない。

ここでは、架橋された骨格筋組織を用いて高品質の核を単離する堅牢な方法について説明します。塵から核を効果的に分離するために逐次濾過を行い、皿状トランスデューサからの超音波エネルギーがChIP分析のためにクロマチンを剪断した。この結果は、標的プロモーターにBMAL1の概日特異的結合を示した。 ChIPは、架橋が起こるときにゲノムDNA上のリアルタイムタンパ...

骨格筋は生理および行動において重要な役割を果たす。多核筋繊維は、アクチンとミオシンが収縮力を発生させる筋節と呼ばれる機能単位を形成する筋原繊維からなる。骨格筋は体内で最大の代謝臓器でもあり、食後のグルコース摂取量が80％を超え、インスリン応答と代謝ホメオスタシスを調節します1,2 。筋肉の生理および代謝は、内在的な時計である内在的な生物学的タイマー3,4,5,6によって厳密に規制されている。例えば、概日の主要な時計成分の1つであるBmal1の骨格筋特異的欠損は、インスリン抵抗性をもたらし、骨格筋におけるグルコース取り込みを減少させ、動物は2型糖尿病を発症することが判明した7 。私さらに、骨格筋も内分泌臓器8として評価され、全身の代謝および生理機能を調節するためにミオシンを分泌する。骨格筋におけるこれらの調節機能を完全に理解するためには、機構的研究が必要である。 ChIPは、DNA結合タンパク質のプロモーター補充を描写するための強力なアプローチである。 ChIPは、最初にクロマチンDNA 9,10上のヌクレオソーム構成を同定するために開発された。ホルムアルデヒド、硫酸ジメチルまたは紫外線照射（UV） 11,12を使用してタンパク質およびクロマチンDNAを架橋する様々な方法が報告されている。ホルムアルデヒド架橋は、最も一般的に使用され、クロマチン構造およびDNA-タンパク質相互作用を保存している9,13,14。架橋クロマトグラフィーinを音波処理により細断し、目的の特定のDNA結合タンパク質15,16に対する抗体で免疫沈降させる。近年、ChIPとNGSを組み合わせたChIP配列決定法（ChIP-seq）は、ゲノム全体の転写因子結合を調べるために開発されており、場合によっては時間経過に伴う動的変化をモニターするために開発されている18,19,20 。例えば、概日ChIP-seq研究は、24時間の概日周期18にわたって時間的に正確な遺伝子発現を駆動する概日クロック成分およびヒストンマーカーのゲノム結合の高度に調整された配列を明らかにした。 最も入手可能なChIPプロトコルは、軟組織（ 例えば、肝臓、脳など ）用に設計されており、骨格タルマシン。繊維が豊富な骨格筋を均質化し、高品質の核21を分離することは技術的に困難であり、特に架橋を必要とするChIP実験の場合には困難である。最近の筋肉ChIP研究22では、衛星細胞を筋繊維から分離し、核を両方の細胞型から、組織消化を含む長期の手順によって調製した。ホルムアルデヒド架橋が単離された核の上で行われる前に、全工程が完了するまでに約3時間を要した。この手順は、筋肉組織をより効率的な均質化に対して不応性にし、高品質の核を生成することができた筋繊維の架橋を回避したが、組織の収集から核の架橋への有意なタイムラグは、 - タンパク質相互作用。対照的に、ほとんどの研究は、リアルタイムDNA-プロテイムを保存するために、実験的処置または組織回収の直後に架橋を行ったn結合12 。架橋前の核単離の第2の欠点は、典型的には3〜4時間間隔で生じる概日検体採取のような時間依存性の適用を排除することである。核を架橋させることなく、解離の直後に単離を進行させる必要があるのに対し、架橋した試料は時間経過全体が完了した後に一緒に処理することができ、実験の一貫性を保証する。 未架橋骨格筋からの核の単離のための他のプロトコールも報告されている。 2つの研究では、勾配超遠心分離を使用して残りの筋原線維および細胞破片から核を分離することが記載されている23,24 。スクロースまたはコロイド勾配超遠心分離が未架橋筋肉組織に有効である一方で、本発明者らの実験は、架橋後、勾配超遠心分離が細胞dから核を分離することができないことを明らかにしたエブリスはグラデーションに乗っています。 したがって、我々は、架橋マウス骨格筋組織を用いて高品質の核を単離するための手順を開発した。グラジエント超遠心分離ではなく、連続したろ過法を考案し、核を残骸から効果的に分離しました。超音波処理後、クロマチンサンプルは、標的プロモーターに結合するBMAL1タンパク質の概日パターンを示すChIP研究に首尾よく適用された。我々の方法は、筋肉組織の様々な機械的研究に広く適用することができる。

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			<td height="20" style="height:20px;width:216px;">Formaldehyde solution</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">F8775&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glycine</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">BP381-5</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Trypan Blue solution (0.4%)</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RNase A</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">10109142001</td>
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			<td height="20" style="height:20px;width:216px;">Protease K</td>
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			<td style="width:288px;">Generated in chicken (Cocalico Biologicals) against antigen aa 318-579, and IgY was affinity purified using the same antigen.&#160;</td>
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			<td height="20" style="height:20px;width:216px;">Chicken IgY Precipitating Resin</td>
			<td style="width:143px;">GenScript</td>
			<td style="width:119px;">L00405</td>
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			<td height="38" style="height:38px;width:216px;">KINEMATICA&#160;Polytron PT2100 Benchtop Homogenizer</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-451-178</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">15mL Dounce tissue grinder</td>
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			<td style="width:119px;">357544</td>
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			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 100&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-19</td>
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			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 70&#181;m</td>
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			<td style="width:119px;">08-771-1</td>
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			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 40&#181;m</td>
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			<td style="width:119px;">08-771-2</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 30 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50030-03</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 20 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50020-03</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 10 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50010-03</td>
			<td></td>
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			<td height="38" style="height:38px;width:216px;">Covaris S2 Focused-ultrasonicator</td>
			<td style="width:143px;">Covaris</td>
			<td style="width:119px;">Model S2</td>
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			<td height="20" style="height:20px;">Labquake&#160;</td>
			<td>Thermo Scientific</td>
			<td>C415110</td>
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			<td height="20" style="height:20px;">CCD Microscope Camera</td>
			<td>&#160;Leica Microsystems</td>
			<td>DFC3000 G</td>
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			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:143px;"></td>
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			<td height="20" style="height:20px;width:216px;">Reagent Kit</td>
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			<td height="20" style="height:20px;width:216px;">DNA extraction kit</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:119px;">K0691</td>
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			<td height="20" style="height:20px;width:216px;">Buffers</td>
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			<td style="width:119px;"></td>
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			<td height="76" style="height:76px;width:216px;">All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation

クロマチン免疫沈降（ChIP）は、クロマチンDNAへのタンパク質結合を決定する強力な方法です。しかしながら、繊維が豊富な骨格筋は、筋原繊維の汚染を最小限に抑えた高品質の核の単離における技術的困難のために、ChIPの課題であった。以前のプロトコールでは、架橋前に核を精製しようと試みたが、これは核形成の延長過程でDNA-タンパク質相互作用の変化のリスクを招く。現在のプロトコールでは、まずマウスから採取した骨格筋組織を架橋し、組織を細かく砕いて超音波処理した。我々は超遠心分離が架橋筋肉組織を用いて筋原線維から核を分離することができないことを発見したので、重要な筋線維汚染のない高品質の核を得るために逐次濾過手順を考案した。その後、超音波装置を用いてクロマチンを調製し、抗BMAL1抗体を用いたChIPアッセイにより、遺伝子プロモーターを標的とするBMAL1のパターン。この濾過プロトコールは、架橋骨格筋組織から高品質核を単離するための簡単に適用可能な方法を構成し、概日および他の時間に敏感な研究のための一貫した試料処理を可能にする。次世代シークエンシング（NGS）と組み合わせて、本発明者らの方法は、骨格筋機能に焦点を当てた様々な機構およびゲノム研究のために配置することができる。

ここでは、リアルタイムDNA-タンパク質相互作用を保存するために、組織回収直後にホルムアルデヒド架橋を行った。しかし、我々は、核分離23,24に一般的に使用されるスクロースまたはコロイド勾配は、筋原線維から核を分離するのに有効でないことを見出した（データ示さず）。その理由は、架橋が核および筋原繊維に同様の重力を与えたためで?...

Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

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

我々は、超遠心分離の必要性を排除した、架橋マウス骨格筋から高品質の核を単離するための濾過ベースのプロトコールを提示し、容易に適用することができる。本発明者らは、核から調製されたクロマチンが、クロマチン免疫沈降およびクロマチン免疫沈降配列決定研究に適していることを示す。

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-filtration-based-method-preparing-high-quality-nuclei-from-cross

Research

JoVE Journal

Biochemistry

8.8K ビュー.  University of Texas Health Science Center at Houston. 我々は、超遠心分離の必要性を排除した、架橋マウス骨格筋から高品質の核を単離するための濾過ベースのプロトコールを提示し、容易に適用することができる。本発明者らは、核から調製されたクロマチンが、クロマチン免疫沈降およびクロマチン免疫沈降配列決定研究に適していることを示す。 

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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

リバーシブルジスルフィド架橋ミセルの生産のための簡便かつ効率的なアプローチ

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

生化学

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

定量的トランスクリプトーム解析に使用するための種子から高度に精製されたRNAの単離のための効率的な方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

単一サンプルからの代謝産物、脂質およびタンパク質の包括的分析のための単純分別抽出法

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

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

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

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

高効率巻き戻しと受容体リガンド結合領域の浄化法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

植物の鉄含有量を測定するための比色定量法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

アフィニティクロマトグラフィー樹脂の急速な発達のための活性化架橋アガロース - ケーススタディとしての抗体捕捉

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

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 ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

高品質発酵魚製品の調製

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

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 ...