この作品は、健康RO1 - GM085130の国立研究所と国立科学財団MCB0929394からの補助金によって支えられている。私たちは、彼女のもてなしのためにと彼女の研究室でフィルムに私たちを可能にするための博士マリオンシュミットに感謝。

1。フットプリントの試薬の調製<ol><li> 100mMのカコジル酸ナトリウム、1mMのEDTA、および1 M KClを含む10 ×反応バッファーを準備します。 7.4にpHを調整する。 0.2μMのアセテートフィルターデバイス（Nalgene）を使用してバッファをフィルタリングする。備考：10 ×バッファに直接ピペットのRNAをしないでください。 </li><li>表1に示すように、各反応の滴定の反応ミックスを調製する。滴定のミックスの音量（所望の濃度で1 ×バッファーとMg（II））が1 ×バッファー中のRNAの10μlを添加する前に、90μlのはずです。 </li><li> 6.63M尿素、20mMのクエン酸ナトリウム、1mMのEDTA、0.25μg/μLのtRNAは、0.025％キシレンシアノール、および0.025％ブロモフェノールブルーを含むリボヌクレアーゼT1消化バッファーを準備します。このバッファは、最大6ヶ月までは4℃で保存することができます。 </li><li>フェントン反応は250mmのFe（NH 4）2（SO 4）2、250mMのEDTA、500 mMのL -アスコルビン酸ナトリウムの新しく調製した水溶液を必要とします。濃度は、第1章で述べた0.5はそのようなラジカルや多様な二価イオンの濃度を清掃しませんカコジル酸ナトリウムなどのバッファーで平衡実験に最適です。 </li><li>過酸化（1.5.1）および酸化（1.5.2）フット反応の試薬。 <ol><li>過酸化フェントン反応は、100 mMのFe - EDTA溶液、及び50mMナトリウムL -アスコルビン酸および1.5％H 2 O 2の新しく調製した水溶液を必要とします。のFe - EDTA溶液は鉄以上のEDTAの1.1倍過剰を持っているEDTAとの混合のFe（NH 4）2（SO 4）2によって調製される。 </li><li>ちょうど酸化的開裂反応（3.4.2）を開始する前に、液のFeの2μlの（NH 4）2（SO 4）2（250 mM）を、EDTA 2.2μL（250 mM）で、62.5を組み合わせることにより、フェントン反応ミックスを作成するL -アスコルビン酸ナトリウム（500 mM）で、22.5μlの10 ×反応バッファーと0のフットプリント反応混合物（3.4.2）での最終濃度に到達するH 2 Oの135.5μL。それぞれ10 mMの、0.11 mMの、および6.61 mMの、。 </li></ol></li></ol> 2。フットプリントの実験用RNAの調製<ol><li> RNAはDNAテンプレート6または、短い50以上のヌクレオチドであれば、購入できる（統合されたDNA技術で例えば、www.idtdna.com）のin vitro転写規格により製造することができる。フットプリント実験中にRNAのサンプルの整合性を維持するために推奨実験室の清浄度の手順に従ってください。7 </li><li> in vitroで転写されたRNAの脱リン酸化8は前のRNAの5&#39;末端の32 P標識に必要です。 RNAの10 pmolのは、末端標識kinasingによるものである。9は、解決策はホスファターゼの成功不活化後曇りになるはず。 </li><li>変性ゲル電気泳動を用いて放射標識RNAを精製する。 RNAは、0.3 M酢酸ナトリウム（pH 5.5）を使用して、続く2つのゲル抽出によって回収される。 RNAは1 mlのエタノールとそれに続くと0.5ミリリットルの水溶液を混合して沈殿させ、ドライアイス上で1分間インキュベーション。 4℃、0.5時間12500 rpmでサンプルをスピン。上清を捨て、70％エタノールでRNAペレットを洗浄。追加の遠心分離後上清を除去し、真空中でペレットを乾燥させます。 </li><li>反応チューブにRNA溶液を、エタノールおよび真空中で乾燥したRNAと沈殿物の1X Buffer.Pipet30μlの330 ULで32 P標識したRNAサンプルを溶かし、リファレンスラダー（ステップ3.5）生成するためにこのチューブを保持。 95℃で加熱することによってバッファリングされたRNA溶液を変性℃で2分間。 15分間室温に試料を冷却し、戻って溶液中にチューブの蓋に凝縮をダウンさせるクイックスピンを与える。 </li></ol> 3。フットプリント実験<ol><li>電気泳動の櫛は、分析ゲルあたりのデータポイントの上限数を決定します。我々は30も櫛を使用してください。リファレンスラダーは2井戸と未切断の制御1つを占有。 27反応チューブを準備します。最後のマグネシウム（II）concentratiアドオンは、均等に滴定の中間点の周りに、桁違いにまたがる対数スケールで等間隔する必要があります。 </li><li>これとは別に、5分間50℃RNAおよびMg（II）ソリューション℃でインキュベートする。 100μlの最終容量に達するまで1 ×バッファー中のMgの対応する量（II）の90μlのRNA溶液の10μlを混ぜる。 50℃で30分のためのソリューションをインキュベート℃に</li><li>折りたたみが行われている間ソリューションは、25℃で1時間平衡化してみましょう。 </li><li>過酸化（3.4.1）または酸化（3.4.2）ヒドロキシルラジカルフットプリンティング反応。 <ol><li> 2μlの鉄- EDTA（100 mm）の液滴を配置することにより、過酸化反応を開始、2μlのL -アスコルビン酸ナトリウム（50mMの）と2μlのH 2 O 2（1.5％）は反応の最内側で互いに分離RNA溶液と激しく混合することによりフットプリント反応を開始するを含むチューブ。 300μlの冷無水エタノールを添加することにより15秒後に反応を停止します。チューブを3-5回回し。沈殿、洗浄しステップ2.4​​のようにペレットを乾燥させます。 </li><li>作りたてのフェントン反応ミックス（1.6）の5μlを添加して酸化的ヒドロキシルラジカルfootprintingreactionを開始します。 25℃で30分間インキュベートするチューブを3-5回回して反応ミックスをクエンチするために冷無水エタノールを300μlを追加。ステップ2.4​​で示したように沈殿物は、ペレットを洗浄し、乾燥させます。 </li></ol></li><li>ステップ1.3で調製した溶液を使用して標準的な手順10によればリボヌクレアーゼT1ダイジェストのリファレンスサンプルを生成する。 </li><li> 8μlのゲルローディング色素II（Ambion社）と確認RNAでRNAペレットを溶解は、ガイガーカウンターを用いて再懸濁されている。 </li><li> 11ロードサンプルつの参照や制御を含む標準的なプロトコールに従って変性、8％ポリアクリルアミドシーケンシングゲルを準備します。 60 Wで別々のRNA断片 - 2.5時間で75 W。一晩貯蔵燐光体スクリーンに乾燥ゲルを公開。たとえば、フィルムレスオートラジオグラフィー用のイメージングシステムと蛍光面をスキャン。ストーム865（GEヘルスケア）や台風（GEヘルスケア）。お使いのコンピュータにゲルのイメージファイルを転送します。 </li></ol> 4。データ解析<ol><li>各ヌクレオチドのヒドロキシルラジカル反応性は、分析用ゲル電気泳動で各バンドの強度を定量することにより決定されます。ダウンロード、インストール、およびオープンSAFA、シングルバンドのフィッティングと定量化のためのオープンソースソフトウェアを使いやすい。12、13のユーザマニュアルの指示に従ってください。簡単に言えば、。ゲルのファイルとしてゲルの画像が続きます。txtファイルとしてRNA配列を読み込みます。バンドの強度を調整する、レーンを定義する、アンカーのレーンを選択し、ゲルの位置合わせを行います。ヌクレオチドのバンドの割り当ては、RNase T1の消化ラダーへの参照で発生します。バンドの積分を定量化し、潜在的に不変残基を正規化して割り当てる正規化/ colorplot機能を使用してください。 。txtファイルとして出力を保存します。 </li><li> SAFAはでを表すゲル上のレーンと行を表す列を含むスプレッドシートを出力するRNA断片に対応する個々のバンドのバンドの密度をtegrated。最初に、エンドポイントのMg（II）濃度（この例での50 mM）のプロファイルを持つマグネシウム（II）のサンプルがないから派生した保護プロファイルを比較することにより、溶媒露出度の顕著な変化を表示する保護サイトを識別する。値が小さいほど、より多くのヌクレオチドは、ヒドロキシルラジカルおよびその逆からの攻撃に対して、保護された。 </li><li>個々の又はMg（II）濃度対ヌクレオチドのグループの正規化されたバンドの積分から折りたたみ等温線を作成します。等温線は、個別にFI = L +（U - L）による飽和度にスケーリングされます• <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> fが分析されているバンド（s）の統合された密度を示す場所、LとUは、移行する下限値と上限値を表し、 <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" />飽和度は14のデータがフィット非線形lを使用しているイーストスクエア解析プログラム（我々は、Origin（OriginLabの）またはグラフパッド（グラフパッドソフトウェア社）のいずれかを使用する。）ヒルの方程式（1）に、 <img alt="式（1）" src="/files/ftp_upload/3244/3244eq1.jpg" /> K dが平衡である解離定数、[M]フォールディング反応を媒介する変数、この例のMg（II）の濃度に相当し、n Hはヒル係数である。この手順のスケール飽和度への移行は、その中間点を決定し、遷移がシグモイドであるかどうかの現象学的テストを提供します。 SAFA（図3B）によって分析し、上記のようにフィットされたシーケンシングゲル（図3A）から派生した等温線を図3Cに示されています。図3に示すように、飽和度の等温線は、スプレッドシートのスケーリングによって生成された数式の小数部を使用して最高のフィット上限値と下限値（UとL）に対してSAFAによって生成された値= 値 /（U - L） - L /（U - L）。 </li></ol> 5。代表的な結果： 図3は、P4 - P6 RNA•OHフットプリント実験から代表的な結果を示しています。ゲルイメージ（左））、バックグラウンドの切断が最小限であること（右車線を示す）、b）単一ヌクレオチドの割り当てが原因T1車線（各GでのRNase T1を切断）で明確に定義されたバンドに可能であり、c）ヒドロキシルラジカルRNAの誘導の断片化は、よくバックグラウンドを超えています。高Mg（II）へのローからの移行は、個人およびRNA立体構造の形成を示すバンドのグループの統合されたバンドの密度を下げると提携しています。シングルまたはその開裂の変化付随して&quot;保護&quot;と呼ばれる連続したヌクレオチドのグループ。密接にその結晶構造に見られる分子の溶媒にアクセスできない領域に対応するマグネシウム（II）の特徴•OHの保護は、P4 - P6 RNAの折りたたみを媒介。15 折られたRNA分子はC<html>明確な構造や動的な現象と相関されていない第三の接点保護の非常に異なるエクステント（つまり、どのバンドの密度の減少の背景に近い）がある。したがって、いくつかのRNA分子は、図3に示すように、明確に定義された構造の遷移が表示され、一部はしません。バンド強度は、SAFA 12解析（図3B）により定量化し、正規化されています。出力は、•OHに対する保護の相対的な度合いを可視化する&quot;熱&quot;のプロットです。色の変化は、Mg（II）を添加すると、アクセシビリティの変化を説明します。それぞれ、赤または青のショーよりアクセス以上の保護されたヌクレオチドの白。シェーディングの各程度は、保護の曲線（図3C）としてプロットし、ヒルの式（1）のような結合モデルによって分析することができる数値と提携しています。保護153から155に所属する平衡解離定数は約2倍のその保護163から164の対応する値のです。 ontent&quot;&gt; <img alt="図1" src="/files/ftp_upload/3244/3244fig1.jpg" /> 図1。ヒドロキシルラジカル生成とP4 - P6 RNAの折りたたみ。 A）のFe（II）は、過酸化水素からヒドロキシルラジカルの生成を触媒する。アスコルビン酸は第一鉄に鉄（III）バックを低減します。 B）マグネシウム（II）の非存在下で、何P4 - P6三次構造は、ヒドロキシルラジカルは、すべてのアクセス可能なバックボーンの位置を貫通して切断できるように、形成されていない。 Mgの加算（II）は、溶媒のみアクセス可能なバックボーンがヒドロキシルラジカルによって切断できるように、P4 - P6の折り畳みが開始されます。 <img alt="図2" src="/files/ftp_upload/3244/3244fig2.jpg" /> 図2。ヒドロキシルラジカルフットプリンティング実験に概要。 ）DephophorylationとRNAの32 P 5&#39;末端ラベリング。 B）変性ポリアクリルアミドゲル上で32 P -ラベルされたRNAの精製。 C）RNAのバンドの切除、その後のRNA抽出、およびエタノール沈殿。 D）PrefoldingとRNAの折りたたみ。 E）新たに調製したフェントン反応混合物の添加は、ヒドロキシルラジカルを生成する。ポリアクリルアミド変性ゲル電気泳動により、F）のRNA断片の分離。 SAFAソフトウェアによるRNA断片のG）定量。 <img alt="図3" src="/files/ftp_upload/3244/3244fig3.jpg" /> 図3。過酸化フェントンフットプリント反応後のRNA断片の解析。 （A）RNAは、ヒドロキシルラジカルにさらさ​​れ、切断産物は変性ポリアクリルアミドゲル電気泳動（PAGE）を用いて解決されました。画像は、Mg（II）の濃度の増加と過酸化開裂生成物を示しています。リファレンスとコントロールレーンが表示されています。このイメージファイルは、各バンドの音量をquantitates SAFAプログラムの入力です。 （B）SAFAによって生成される&quot;熱&quot;プロット。 （C）SAFAからヌクレオチド数の出力に統合されたバンドの密度を関連する値のスプレッドシートは、ヒルequatiのような結合モデルによって分析され（1）。保護地域は、Mg（II）濃度の関数としてバンドの積分密度の増加に応じて選ばれた。 K dは、RNAの半分が監視されているサイトで折り畳まれる時にマグネシウム（II）濃度である。ヒル係数、n Hは 、バインディングの共同性に関する情報を提供し、この特定のサイトのフォールディングに関与するマグネシウムイオンの数の下限推定値を提供する曲線の傾きの尺度です。 <img alt="表1" src="/files/ftp_upload/3244/3244table1.jpg" /> 表1。異なるマグネシウム（II）濃度を含むRNAサンプルを生成するための代表的なボリューム。

<ol>
	<li>Tullius, T. D., Dombroski, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl+radical+"footprinting":+high-resolution+information+about+DNA-protein+contacts+and+application+to+lambda+repressor+and+Cro+protein.">Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 5469-5473 (1986).</li><li>Celander, D. W., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron(II)-ethylenediaminetetraacetic+acid+catalyzed+cleavage+of+RNA+and+DNA+oligonucleotides:+similar+reactivity+toward+single-+and+double-stranded+forms.">Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: similar reactivity toward single- and double-stranded forms.</a> Biochemistry. 29, 1355-1361 (1990).</li><li>Celander, D. W., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+higher+order+folding+of+a+catalytic+RNA+molecule.">Visualizing the higher order folding of a catalytic RNA molecule.</a> Science. 251, 401-407 (1991).</li><li>Sclavi, B., Sullivan, M., Chance, M. R., Brenowitz, M., Woodson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+folding+at+millisecond+intervals+by+synchrotron+hydroxyl+radical+footprinting.">RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting.</a> Science. 279, 1940-1943 (1998).</li><li>Tullius, T. D., Dombroski, B. A., Churchill, M., Kam, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl+radical+footprinting:+a+high-resolution+method+for+mapping+protein-DNA+contacts.">Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts.</a> Methods. Enzym. 155, 537-558 (1987).</li><li>Milligan, J. F., Groebe, D. R., Witherell, G. W., Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligoribonucleotide+synthesis+using+T7+RNA+polymerase+and+synthetic+DNA+templates.">Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.</a> Nucleic. Acids. Res. 15, 8783-8798 (1987).</li><li>Schlatterer, J. C., Brenowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementing+global+measures+of+RNA+folding+with+local+reports+of+backbone+solvent+accessibility+by+time+resolved+hydroxyl+radical+footprinting.">Complementing global measures of RNA folding with local reports of backbone solvent accessibility by time resolved hydroxyl radical footprinting.</a> Methods. 49, 142-147 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (2001).</li><li>Zaug, A. J., Grosshans, C. A., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+endoribonuclease+activity+of+the+Tetrahymena+ribozyme:+enhanced+cleavage+of+certain+oligonucleotide+substrates+that+form+mismatched+ribozyme-substrate+complexes.">Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes.</a> Biochemistry. 27, 8924-8931 (1988).</li><li>Knapp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+approaches+to+probing+of+RNA+secondary+and+tertiary+structure.">Enzymatic approaches to probing of RNA secondary and tertiary structure.</a> Methods. Enzym. 180, 192-212 (1989).</li><li>Slatko, B. E., Albright, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+gel+electrophoresis+for+sequencing.">Denaturing gel electrophoresis for sequencing.</a> Curr. Protoc. Mol. Biol. Chapter 7, Unit 7.6-Unit 7.6 (2001).</li><li>Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., Altman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAFA:+semi-automated+footprinting+analysis+software+for+high-throughput+quantification+of+nucleic+acid+footprinting+experiments.">SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments.</a> RNA. 11, 344-354 (2005).</li><li>Simmons, K., Martin, J. S., Shcherbakova, I., Laederach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+and+analysis+of+kinetic+*OH+radical+footprinting+data+using+SAFA.">Rapid quantification and analysis of kinetic *OH radical footprinting data using SAFA.</a> Methods. Enzym. 468, 47-66 (2009).</li><li>Uchida, T., He, Q., Ralston, C. Y., Brenowitz, M., Chance, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+of+monovalent+and+divalent+ion+binding+in+the+folding+of+the+P4-P6+domain+of+the+Tetrahymena+ribozyme.">Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme.</a> Biochemistry. 41, 5799-5806 (2002).</li><li>Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+group+I+ribozyme+domain:+principles+of+RNA+packing.">Crystal structure of a group I ribozyme domain: principles of RNA packing.</a> Science. 273, 1678-1685 (1996).</li><li>Petri, V., Brenowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+nucleic+acids+footprinting:+thermodynamic+and+kinetic+approaches.">Quantitative nucleic acids footprinting: thermodynamic and kinetic approaches.</a> Curr. Opin. Biotechnol. 8, 36-44 (1997).</li><li>Takamoto, K., Das, R., He, Q., Doniach, S., Brenowitz, M., Herschlag, D., Chance, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+RNA+compaction:+insights+from+the+equilibrium+folding+pathway+of+the+P4-P6+RNA+domain+in+monovalent+cations.">Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations.</a> J. Mol. Biol. 343, 1195-1206 (2004).</li><li>Brenowitz, M., Senear, D. F., Shea, M. A., Ackers, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+DNase+footprint+titration:+a+method+for+studying+protein-DNA+interactions.">Quantitative DNase footprint titration: a method for studying protein-DNA interactions.</a> Methods. Enzym. 130, 132-181 (1986).</li><li>Shcherbakova, I., Mitra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl-radical+footprinting+to+probe+equilibrium+changes+in+RNA+tertiary+structure.">Hydroxyl-radical footprinting to probe equilibrium changes in RNA tertiary structure.</a> Methods. Enzym. 468, 31-46 (2009).</li><li>Howlett, G. J., Minton, A. P., Rivas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+ultracentrifugation+for+the+study+of+protein+association+and+assembly.">Analytical ultracentrifugation for the study of protein association and assembly.</a> Curr. Opinion. Chem. Biol. 10, 430-436 (2006).</li><li>McGinnis, J. L., Duncan, C. D., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+SHAPE+and+hydroxyl+radical+analysis+of+RNA+structure+and+ribonucleoprotein+assembly.">High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly.</a> Methods. Enzym. 468, 67-89 (2009).</li><li>Jonikas, M. A., Radmer, R. J., Laederach, A., Das, R., Pearlman, S., Herschlag, D., Altman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+modeling+of+large+RNA+molecules+with+knowledge-based+potentials+and+structural+filters.">Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters.</a> RNA. 15, 189-199 (2009).</li></ol>

利害の衝突は宣言されません。

ヒドロキシルラジカルフットプリントは、核酸の溶媒接触可能表面積を評価する貴重なツールです。三次構造14の定性的および定量的な形成は、イオンの種類や濃度、pH、温度、結合タンパク質または折りたたみ共同因子としてのパラメータの関数として追跡することができる。まっすぐ進むと安価なプロトコルの強力な組み合わせと単一ヌクレオチドレベルで、その結果、溶媒アク?...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA分子は、生物学において重要な役割を果たしている。遺伝情報を伝達することに加えて、RNAはレギュレータ、バインダーや触媒などの特定の生物学的役割を果たすユニークな立体構造に折りたたまれることができる。三次接触の形成についての情報は、RNA分子の機能を理解することが不可欠です。ヒドロキシルラジカル（•OH）は、その高い反応性と小さなサイズのため、核酸の構造のユニークなプローブである。1フットのプローブとして使用する場合は、ヒドロキシルラジカルがDNAを1とを有するRNA 2のホスホジエステルバックボーンの溶媒接触可能表面をマッピング一塩基の解像度と同じくらい素晴らしい。ヒドロキシルラジカルフットプリンティングはDNA -タンパク質1とRNA -タンパク質複合体で、例えば、分子間の接触面内のヌクレオチドを識別するために使用することができます。平衡3と4の遷移運動は、solutiの関数としてヒドロキシルラジカルフットプリンティングを行うことによって決定することができます変数または時間で、それぞれ。フットプリントの主な特徴は、プローブにその限られた暴露（例えば、&quot;シングルヒット動力学&quot;）ポリマーの各ヌクレオチドの均一サンプリングの結果です。5 このビデオの記事では、我々は、RNAサンプルの調製およびMg（II）を介したフォールディング等温線の測定を説明するために、 テトラヒメナのリボザイムのP4 - P6のドメインを使用してください。我々は、H 2 O 2（我々はこの&quot;過酸化&quot;プロトコルと呼ぶ）と自然に溶存O 2を使用している貴重な、しかし広く知られていない、別のを（我々が呼ぶこの&quot;必要とするよく知られているヒドロキシルラジカルフットプリンティングプロトコルの使用を説明し酸化&#39;プロトコル）。データの削減、変換および分析手順の概要が提示される。

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

このプロトコルは、Mg（II）依存性ヒドロキシルラジカルフットプリンティングの2つの方法でRNA三次構造の形成を定量化する方法について説明します。

&quot;過酸化&quot;と&quot;酸化&quot;ヒドロキシルラジカルフットプリントにより、RNAの構造における均衡の変化を監視する

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

monitoring-equilibrium-changes-rna-structure-peroxidative-oxidative

Research

JoVE Journal

Biology

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

14.2K ビュー.  Hunter College. このプロトコルは、Mg（II）依存性ヒドロキシルラジカルフットプリンティングの2つの方法でRNA三次構造の形成を定量化する方法について説明します。

ビデオ: Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

二層弾性特性の変化をモニタリングするための単一分子法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

生物学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

蛍光によるウイルスRNAの検出<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

ハイスループットSHAPEを使用したRNA二次構造予測

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Colocalizational解析による受容体後の内在トラフィッキングにおける薬物誘発性の変更の追跡

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...