この作品は、カナダのヘルスリサーチの研究所（CIHR）、国立科学と工学研究評議会（NSERC）、およびCIHR訓練助成金（LFへ）によってサポートされていました。私たちは、AACのための教授ジェラルドD.ライト（マクマスター大学、カナダ）（6）- IIの発現は、プラスミド感謝。

1。ストック溶液を準備する<ol><li>興味のある高分子を精製する。 （このケースでは、アミノ配糖体N - 6&#39; -アセチルトランスフェラーゼ- II（AAC6&#39; - II）は、他の場所で報告されたとして単離される。13） </li><li>透析バッファー4リットルを準備します。 （このケースでは、25mMの4使用 - （2 - ヒドロキシエチル）-1 - piperazineethanesulfonic酸（HEPES、MW 238.3グラム/モル）、2 mMのエチレンジアミン四酢酸（EDTA、MW 292.2）を含む、pH7.5で。） </li><li>タンパク質を透析に使用AAC（6&#39;）- IIのサンプル（400μMで5mL）を透析緩衝液（3 × 1.3 L）中で透析する必要があります。最終的な透析液は、機械を洗浄するとサンプルを希釈するために保持されます。 </li><li> 0.45μmのセルロースフィルターを通して最終的な透析液をフィルタリングし、4℃で保存&quot;実行中のバッファ&quot;と呼ぶことにする</li><li>実行中のバッファで十分にリンスされている0.2μmのシリンジフィルターを通してフィルタータンパク質溶液。標準アッセイ（Bradford、Lowry法、等）またはUV吸光度を用いてタンパク質の最終濃度を測定する。長期的な安定性を最大化するタンパク質を保管してください。 （AAC（6の場合には&quot;）- II、4でこの手段の記憶℃、AAC（6&#39;）- IIは、凍結融解後の活性を保持していません）。 </li><li>バッファを実行するには4.0 mgを溶解することによってアセチル補酵素Aの25mMのストック溶液（AcCoA、リガンド、MW 809.57）の200μLを準備します。 -78℃で凍結℃で使用可能な状態になるまで。 </li><li>すべての蛋白質とリガンドのサンプルは、濃度のランダムなサンプル間の変動を最小限に抑えるために、同じストック溶液から発信される必要があります。全体の変数に- cデータセット間のタンパク質濃度に対して1つの補正係数は、解析に調整されます。10 </li></ol> 2。準備ITCサンプル<ol><li> 2 mLの最終容積で64のcの値に酵素溶液を希釈する。 （AAC（6&#39;）- IIの場合には、これは192μMに相当する。） </li><li>すぐに氷水のリガンド（AcCoA）ストック溶液を解凍。 </li><li> 10倍以上のAcCoA溶液0.5 mLを準備するバッファを実行して、420μLでAcCoAストック溶液80μLを希釈することにより、この場合は4 mMの、蛋白質にして結合部位の数を濃縮する。ピペット充填チューブに移す。すぐに-78℃にAcCoAのストック溶液を返す</li><li>ドガ温度1で5分間真空下でタンパク質とソリューション℃の希望実行されている温度（19℃）以下。 </li></ol> 3。注射器14の設定<ol><li>あらかじめ実装されているシリンジクランプをホルダーと同じ高さになるまでシリンジホルダーを介して注射器を挿入します。 </li><li>それがしっかりとシリンジホルダーの底部が押し付けられるまで注射器を介して別のシリンジクランプを養う。静かに提供さ0.050&quot;ボールポイント六角ドライブ付きクランプを締めます。 </li><li>ピペットホルダーにシリンジホルダーを置きます。 </li><li>ゆっくりと注射筒にピペットインジェクターをスライドさせます。プランジ​​ャーの先端が注射器の穴に直接供給されていることを確認します。一度完全に挿入、ピペットインジェクタにシリンジホルダーのロッキングカラーをねじ込みます。 </li></ol> 4。試料セル14をロード<ol><li>バッファを実行しているのを50mLの最小で試料セルを洗浄して、そして長期ニードル2.5 mlのガラス製注射器を使用して、残りの液体を取り除く。 </li><li>徐々に清潔で乾燥した長期ニードル2.5 mlのシリンジへのタンパク質の試料溶液1.8 mlの最小値を引く。任意の気泡を導入しないように注意してください。 </li><li>慎重に試料セルに針を挿入し、ゆっくりと、セルの底部に触れない。少しヒントを上げる（〜1 mm）と過剰の液体が試料セルの最上部より上に見えるようになるまで穏やかに細胞内にAAC（6&#39;）- II溶液を注入する。 </li><li>オーバーフローの液体遺跡を確保しつつ、ゆっくりと約1cmの針を上げる。すぐに撤回し、試料セル内の任意の閉じ込められた気泡を除去するために少量の溶液（〜0.25 ml）を注入する。 </li><li>すべてのソリューションのオーバーフローを削除します。これは、静かにサンプルセル中にオーバーフローの側面に沿って針をスライドさせることにより達成される。注射器の先端が棚にヒットする、これは実行中のソリューションのための欲求の高さです。この行の上に座っているすべての液体を削除します。 </li></ol> 5。注射器のロードと実行14を開始する<ol><li>充填ポートにプラスチック製のチューブローディングシリンジを取り付けます。 </li><li>充填ポートの上部にプランジャーの先端を下げます。 </li><li>ピペットホルダーの底部にピペット充填チューブにAcCoAソリューションを置きます。シリンジの先端は、充填チューブの底に触れないでください。 </li><li>少量のローディングシリンジのチューブに入るまで、ゆっくりとシリンジに解決策を描く。 </li><li>プランジ​​ャーの先端を下げることによって、充填ポートを閉じ、[閉じる充填ポートのボタンをクリックしてください。ローディング中に閉じ込められる可能性のあるすべての気泡を除去するパージ - &gt;リフィルのボタン、3回をクリックしてパージし、詰め替え、。 </li><li>ピペットホルダーから充填チューブを外し、ゆっくりとシリンジの先端を拭きます。 </li><li>ピペットのアセンブリおよび試料セルに優しく低注射器を取る。ゆっくりとシリンジを簡単に曲げることができるように実行しますので、細心の注意が必要です。注射器が完全に襟のロックの基盤を押し下げて、挿入されていることを確認します。 </li><li>希望する実行中の温度を（この場合20℃で）設定し、予想される最大射出熱流量（この場合の20μcal/secで）よりも若干大きくなりますので、基準電源を選択する。 </li><li>希望の注入量と遅延をプログラムする。 （この場合は、28注射が使用された。最初の注射は60秒遅れで2μLの容積を持っていた。後続のすべての注射は、330秒の遅延と10μLのボリュームを持っていた。） </li></ol> 6。以降の実行<ol><li> 2,4,8,16、および32倍にAAC（6&#39;）- IIとAcCoAの濃度を低減しつつ、すべての後続の実行では、手順2〜5を繰り返します。 </li></ol> 7。データ解析<ol><li>世界的に前述の10に記載の結合パラメータの単一のセットに等温線のすべてに適合するフィッティングの手順を使用します。 </li></ol> 8。代表的な結果代表的なデータを図1に示されています。等温線の形状は、濃度によって変化する必要があります。シャープな遷移は高いが期待されているC -値（すなわち、より高い蛋白質とリガンドの濃度）（図2）。 AAC（6&#39;）- IIの場合、2サイトシーケンシャルモデルでは、調整可能な化学量論と同一の、独立したサイトの2つのセットを記述するよりも優れたフィット感を与えます。 <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="図1" /> 図1。AACにAcCoA（3.86 mM）の（6&#39;）- II（192μM）の滴定によって生成される等温線。 A）生ITCトレース。 B）2サイトシーケンシャルフィット（との結合パラメータを（正方形）を決定するために使用される統合された値が - ）。 <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="図2" /> AcCoAについては図2。ITCの等温線は、様々な濃度でAAC（6&#39;）- IIに滴定する。実験データ（白丸）を2セット - の部位に依存しないモデル（破線マゼンタ）と2 - サイトシーケンシャルモデル（ソリッドブルー）に適合させた。 2サイトsequentionalモデルは明らかに良い全体的な合意が得られます。採用濃度は）6μM、0.25 mMの、B）12μM、0.25 mMの、C）24μM、0.5mMの、D）48μM、1.0mMの、E）96μM、1.9 mMの、およびF）196μM、 AAC（6&#39;）- IIとそれぞれAcCoAのための3.86 mMの、。

<ol>
	<li>Cliff, M. J., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <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+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Mechanism+of+the+GCN5-Related+Chromosomal+Aminoglycoside+Acetyltransferase+AAC(6')-Ii+from+Enterococcus+faecium:+Evidence+of+Dimer+Subunit+Cooperativity.">Kinetic Mechanism of the GCN5-Related Chromosomal Aminoglycoside Acetyltransferase AAC(6')-Ii from Enterococcus faecium: Evidence of Dimer Subunit Cooperativity.</a> Biochemistry. 42, 6565-6574 (2003).</li><li>Wright, G. D., Ladak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+characterization+of+the+chromosomal+aminoglycoside+6'-N-acetyltransferase+from+Enterococcus+faecium.">Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.</a> Antimicrob. Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

利害の衝突は宣言されません。

変数に- cフィッティングのこの分析部分は、以前は詳細10で説明されています。ここでは、このアプローチに適した変数に- cデータセットを収集するの実用的側面を報告する。それはすべての蛋白質とリガンドのサンプルが同じストック溶液から引き出されていることが不可欠です。したがって、十分なストック溶液は実験のシリーズ全体を完了するために最初に用意されていること...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
</tr>		</tbody>
		</table>
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collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

等温滴定熱量測定（ITC）は、一般的に、ホスト高分子のリガンドの結合に関連する熱力学的パラメータを決定するために使用されます。 ITCは、ホスト/リガンド相互作用を研究するための一般的な分光学的方法に比べていくつかの利点があります。たとえば、2つのコンポーネントが相互作用するときに放出または吸収された熱が直接測定され、任意の外生的な記者を必要としません。したがって、結合エンタルピーと会合定数（KA）は直接ITCデータから得られる、とエントロピーの寄与を計算するために使用することができます。また、等温線の形状は、c -値と関係する機械的なモデルに依存しています。 C -値は、[P] Tはタンパク質濃度であり、そしてnは、ホスト内のリガンド結合部位の数であり、C = N [P] TKA、として定義されています。多くの場合、特定のリガンドのための複数の結合部位は非等価であり、ITCは、個々の結合部位の熱力学バインディングパラメータの特性評価が可能になります。しかし、これは正しいバインディングモデルを使用する必要があります。異なるモデルで同じ実験データをフィットできる場合は、この選択は、問題となる可能性があります。我々は以前この問題は、c -値のいくつかで実験を行うことにより回避できることが示されている。 C -値は異なるで得られた複数の等温線は、別のモデルを同時にフィットされています。正しいモデルは、次の変数全体の- cデータセット全体の適合度に基づいて識別されます。このプロセスは、アミノグリコシド耐性の原因となる酵素アミノグリコシドN - 6&#39; -アセチルトランスフェラーゼ- II（AAC（6&#39;）- II）にここに適用されます。私たちの方法論は、あらゆるシステムに適用可能であるが、この戦略の必要性が良くアロステリック効果や協同性を示す高分子 - リガンドシステムで実証、および別の結合モデルは、同じデータに本質的に同一のフィットを提供する場合です。私たちの知識に、市販のそのようなシステムはありません。 AAC（6&#39;）- II、二つのサブユニット間の協同性を示す、二つの活性部位を含むホモ二量体である。しかしC -値の単一で得られたITCのデータは、少なくとも2つの異なるモデルが二組 - の部位に依存しないモデルと二サイトシーケンシャル（協同組合）のモデルにも同様にフィットすることができます。上記で説明したように、C -値を変化させることを通じて、それは、AACの正しい結合モデル（6&#39;）- IIは、2つのサイトシーケンシャル結合モデルであることが設立されました。ここで、我々は、ITCの実験を行う際に、変数- cの分析に適したデータセットを得るためにに必要な手順を説明します。

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

ITCは、そのホストへのリガンドの結合を研究するための強力なツールです。複雑なシステムではしかし、いくつかのモデルも同様にデータをフィットすることがあります。ここで説明する方法は、複雑なシステムのための適切な結合モデルを解明し、対応する熱力学的パラメータを抽出する手段を提供します。

バインディング機構を決定するために、可変濃度等温滴定熱量計のデータセットの収集

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

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JoVE Journal

Biology

12.7K ビュー.  McGill University. ITCは、そのホストへのリガンドの結合を研究するための強力なツールです。複雑なシステムではしかし、いくつかのモデルも同様にデータをフィットすることがあります。ここで説明する方法は、複雑なシステムのための適切な結合モデルを解明し、対応する熱力学的パラメータを抽出する手段を提供します。

ビデオ: Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

免疫ペルオキシダーゼアッセイを用いたヒトコロナウイルスの滴定

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

生物学

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

手術創における侵害受容性および炎症性メディエーターを収集し、測定

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

プロテアーゼ活性を測定するためにプロテアーゼ蛍光検出キットの使用

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR -クリップ - RNA結合タンパク質のトランスクリプトーム全体の結合部位を同定する方法

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

高分子 - リガンド親和性を測定するための等温滴定カロリメトリー

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

管細胞の収集培養初代ラットインナー髄

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

グリコーゲンの生化学的滴定<em&gt;インビトロ</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

転写開始点を決定するための蛍光に基づくプライマー伸長技術とのRNaseの切断サイト<em&gt;インビボ</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

外酸生産の測定と解析は、解糖速度を決定するために

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.