著者認めるコスト アクション CM1406 (エピジェネティックな化学生物学)。この出版物で報告された研究は、ピエール ・ メルシエ財団によって支えられました。

1. 細胞培養
注: 次の手順は、標準のティッシュ文化フードで実行されます。生殖不能の技術に精通するいるとされます。
<ol>
	<li>10% 牛胎児血清ペニシリン G を添加した MEM でコート t75 フラスコ細胞培養用フラスコの 80% の confluency に HeLa 細胞の培養 (100 U/mL) とストレプトマイシン (100 mg/mL)。 注: セル文化、治療、および換散の手順は 5% CO2無菌条件下での加湿雰囲気で 37 ° C で細胞培養手順を伴う層流の下で行われます。</li>
	<li>DPBS 5 mL で 2 回洗浄、吸引、DPBS 細胞解離試薬 1 mL を加えます。セルをデタッチする 5 分の 37 ° C で孵化させなさい。</li>
	<li>MEM 培地の 9 mL を追加し、徹底的に細胞懸濁液を再懸濁します、診断を使用してセルをカウントします。</li>
	<li>6 × 104セル/ml MEM に細胞懸濁液の密度を調整します。</li>
	<li>コントロールに細胞懸濁液 100 μ L を追加し、(図 1 a)、滅菌、平底、組織培養治療 96 ウェル プレートの井戸をテストします。</li>
	<li>96 ウェル プレート (図 1 a) の空井戸に MEM 培地の 100 μ L を追加します。</li>
	<li>24 h の 37 ° C で 96 ウェル プレートを孵化させなさい。 注: 24 時間培養は、HeLa 細胞の内因性 HDAC アクティビティをテストする適切な時間です。不明な HDAC 活動と異なるセル型は、制御の細胞の経時的評価を必要があります。</li>
</ol>
2 テスト混合物と HDAC 基板細胞治療
<ol>
	<li>96 ウェル プレートの井戸から培地を吸引します。</li>
	<li>テスト混合物 x 2 を 25 μ l 添加(例えば、トリコスタチン A、MS275、tubastatin、) 井戸をテストする MEM で。コントロールと空白の井戸に MEM の 25 μ L を追加します。 注: テスト混合物の濃度は、少なくとも 5 の最終的な試験濃度 (ウェルあたり 1 つ濃度) の範囲を提供するために調整することです。化合物のテストで DMSO 最終濃度 0.5% を超えないようにする必要があります、空白やコントロールを含む、各ウェルで同じである必要があります。最終的なインキュベーション ボリュームは、ウェルあたり 50 μ L です。トリコスタチン A は 500 から 1.95 に至るまで 5 の濃度テスト nM (1:4 希釈)。6.17 8,000 に至る 5 濃度テストする MS275 と tubastatin A nM (希釈 1:6)。各々 のユーザーが目的の最終的なテスト各試験化合物濃度を決定して用量反応曲線を作成してください。</li>
	<li>コントロールとテスト井戸に基板の混合物 (マル、MOCPAC の 42 μ M と MEM で BATCP の 42 μ M 42 μ M) の 25 μ L を追加します。空井戸に MEM の 25 μ L を追加します。 注: 各基板の最終濃度は 21 μ M. MOCPAC と BATCP がそれぞれ HDAC1 と HDAC6 の活動の区別 (図 1 b)。エピウエハーと活動は認められなかったと、マルの使用可能性別 HDAC アイソ フォームに対する活動の id。</li>
	<li>加湿 5% で 8 h の 37 ° C で 96 ウェル プレートを孵化させなさい CO2インキュベーター。 注: このインキュベーション時間は HeLa 細胞に適しており、合成の基質と対話する内因性 HDAC をことができます。細胞は培養時間および/または基板濃度の調整を必要があります。</li>
</ol>
3. 細胞換散およびクロマト ・ MS/MS のサンプル準備
注意: サンプルの準備の手順は非常に可燃性、摂取または吸入によって有害有機化学物質を使用します。呼吸用保護具、手袋、保護メガネなどを着用してください。
<ol>
	<li>X RIPA バッファーを 6 (10 x RIPA バッファーから希釈) 96 ウェル プレートの各ウェルにプロテアーゼ阻害剤を添加したの 10 μ L を追加します。</li>
	<li>冷たいアセトニ トリルの 160 μ L を各ウェルに追加によって反作用を停止し、上下にピペッティングで混ぜます。 注: この手順の前に-20 ° C で、アセトニ トリルを続けます。</li>
	<li>10 分間-80 ° C のフリーザーにプレートを配置します。</li>
	<li>冷凍庫からプレートを削除し、各ウェルの内容の 220 μ L を生殖不能、円錐底 (V 下) 96 ウェル プレートに転送します。5,000 × g と 10 分のための 4 ° C でプレートを遠心します。 注: この手順は、蛋白質および塩の取り外しを目指しています。必要に応じて、ソリューションを遠心分離前に個々 の円錐形遠心チューブに転送または 0.65 μ m PVDF フィルター プレート 96 ウェルのセットをフィルター処理できます。沈殿物の成功の除去は、クロマト分析の前に必要です。</li>
	<li>UHPLC システムと互換性のある 96 ウェル プレートに上清 (または濾液) の 200 μ L を転送し、プレートのシーラーを使用して剥離、ヒートシールの箔シールします。 メモ: は、培養上清を削除する場合は、ペレットをピペッティングしないでください。クロマト分析を即座に実行できない場合は、分析まで 24 h 最大 4 ° C でプレートに格納できます。</li>
</ol>
4. UHPLC/MS-質量分析
<ol>
	<li>移動相システム (95% A:5 %b) で塗りつぶすことにより UHPLC システムを準備します。 A: H2O、HPLC グレード 0.1% ギ酸を含む (1 L を準備) B: アセトニ トリル、HPLC グレード, 0.1% ギ酸を含む (1 L の準備)。 注意: 移動相には、非常に可燃性、摂取または吸入によって有毒な有機化学物質が含まれています。呼吸用保護具、手袋、保護メガネなどを着用してください。</li>
	<li>96 ウェル プレートは、プレート ホルダーを含むサンプル マネージャーに。表 1にクロマト ・ MS/MS 条件に従ってサンプルを実行します。 注: 分析の 96 ウェル プレートのすべての列も、よく 1 つのコントロールと 1 つの空白も (図 1 aのプレート方式で描かれている) です。コントロールが、ブランクがある MS ソースは一貫性のある背景を提供するかどうかを確認するテスト細胞の酵素活性を 100% の代表。指定された空白で検出された異常な MS ピークスは、MS 感度変更を防ぐために調べる必要があります。</li>
</ol>
5. データの解析
<ol>
	<li>各基板とそのされたきちん製品のピーク面積を適切なの UHPLC 統合ソフトウェアに統合します。 注: は、平滑化手法 (平均、ウィンドウ サイズ 3 と 2 を例えば滑らかの数) と自動統合パラメーターを使用します。表1 クロマトグラフィー条件を使用して、溶出順序は、dMAL (5.8 分)、dBATCP (6.0 分)、dMOCPAC (6.2 分)、マル (7.6 分)、MOCPAC (8.9 分)、BATCP (9.8 分)、図 1に示されています。</li>
	<li>各基板 (されたきちん/アセチル化) (テストおよび制御サンプル) 各クロマト グラムのピーク面積比を計算します。</li>
	<li>各テスト サンプルのパーセント HDAC 阻害を計算します。 
	<img alt="Equation" src="/files/ftp_upload/55878/55878eq1.jpg"> 注: HDAC1 阻害は、dMOCPAC/MOCPAC; のピーク面積比を使用して、計算します。HDAC6 阻害は、dBATCP/BATCP と計算されます。比 dMAL/マルは、一般的な HDAC 阻害を表現する使用できます。</li>
</ol>

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著者が明らかに何もありません。

HDAC 阻害は、がん療法21HDAC6 阻害の現在のフォーカスの創薬におけるホットな話題です。HDAC の選択性は通常個々 HDAC アイソ フォーム21に向けて抑制効力を判断する高スループット、携帯無料の試金のシリーズによって評価されます。しかし、阻害の選択性またによって確認されなければならない細胞のヒストンなどの内因性のタンパク質の基質のアセチル?...

Hdacs をしたはクロマチン構造中のヒストンを deacetylate することができる酵素の家族に属しています。また、細胞質で他の蛋白質の基質がある、細胞の様々 な区分であります。18 HDAC アイソ フォームの合計これまで同定されている細胞シグナル伝達し同様に、転写因子や遺伝子発現の調節など、いくつかの細胞メカニズムに関連しているし、1,2,3,4,5,6,7を輸送します。触媒の HDAC 阻害剤はがん治療のための潜在的な治療薬として浮上しています。ほとんど HDAC の抑制剤は、現在 FDA によって承認されて T 細胞リンパ腫、多発性骨髄腫の治療のため、ボリノスタット (サハ)、belinostat、パノビノスタット8,9などの非特異的 HDAC 阻害剤です。しかし、副作用のシリーズはパン阻害薬に関連付けられている、アイソ フォーム特異的小さな分子の検索は、薬効がある化学および薬剤の発見のホットな話題。したがって、クラス私 (HDAC1 3 と見られる HDAC8) の選択的阻害剤ロミデプシンは既に承認された薬10特定の HDAC6 阻害剤は、現在多発性骨髄腫11,12,13,14,15に高められた治療の可能性と、臨床試験中。
スクリーニング アッセイ HDAC を特徴づける阻害剤は酵素ソース (1 つのアイソ フォーム、核エキスまたは細胞ライセート) と HDAC 基板のインキュベーションに基づいています。基材は通常 N-(4-methyl-7-aminocoumarinyl)-Nα-(t-butoxycarbonyl)-Nω-acetyllysineamide (マル)16など分解蛍光体 (例えばクマリン) と結合したアセチル リジン残基を含む小さいペプチッド シーケンスです。アイソ フォーム固有のアクティビティを区別するために各アイソ フォームを含む別の無料のセル アッセイが必要と細胞内本物のアイソ フォームの活動を反映していないこと。アイソ フォーム固有の基板ベンジルなど、市販されている (S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate (MOCPAC、HDAC1 特異的基質) と (S)-[5-acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamic 酸 tert-ブチル エステル (BATCP、HDAC6 特異的基質) (図 1 b)。ただし、マルを含む複数基質混合物、MOCPAC、および BATCP 細胞に与えられるを許可しません個々 されたきちん製品の検出蛍光測定による同じ分解蛍光体を抱くことを考える。
ここで説明する方法は、検出と各基板とそのされたきちん製品 UHPLC-ESI ・ MS/MS 分析17続いてマルチ基板アッセイを用いた HeLa 細胞での相対的な定量化のためことができます。HDAC 阻害活性と内因性 hdacs をした試験化合物の特異性の直接同定法を有効に HeLa 細胞に HDAC アッセイを行ったHDAC1 と HDAC6 が同時に評価の焦点があります。単一培養の試金でこれらの酵素の測定を達成するため、96 ウェル プレートにメッキ処理と未処理の HeLa 細胞を非特異的, 特異 HDAC の基板上の混合物を追加します。インキュベーションのステップでは、次のセルは基板と区切られ、UHPLC-質量分析法 (図 1) を使用して検出された彼らのそれぞれの反応製品を解放する服従に分離します。マル、MOCPAC、および BATCP の基板のされたきちんの製品は、それぞれされたきちん MAL (dMAL)、されたきちん MOCPAC (dMOCPAC) とされたきちん BATCP (dBATCP) です。用量反応曲線は、活性化合物を構築できます。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55878/55878fig1.jpg"> 
図 1: 複数基板の UHPLC MS 解析による HDAC1 固有、HDAC6 阻害剤を識別するために細胞を用いた HDAC 試金のための一般的な方式です。(A) 方式、標準の 96 ウェル プレート治療 (試験化合物を含む) の (無処理) HeLa 細胞と無細胞ブランク。(B) 混合物 (各 21 μ M) として内因性 hdacs をした、されたきちんするのに追加基板の化学構造。(C) (マル、MOCPAC、および BATCP) の追加基板とされたきちん製品のピークを示す典型的なクロマト ・ MS クロマト グラム (dMAL、dMOCPAC と dBACTP、それぞれ)。<a href="http://ecsource.jove.com/files/ftp_upload/55878/55878fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BATCP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">B4061</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAL</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>SCP0168</td>
			<td>Synonym: BOC-Ac-Lys-AMC&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MOCPAC</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>M2195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MS275</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">EPS002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trichostatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">T8552</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tubastatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">SML0044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acetonitrile, HPLC grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10660131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formic acid, LC/MS grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10596814</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:271px;">H2O, HPLC grade</td>
			<td style="width:191px;"></td>
			<td style="width:152px;"></td>
			<td>distilled H2O filtered through a Milli-Q purification system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">HeLa cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:152px;">ATCC CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell dissociation reagent TrypLE Express</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">12604013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMSO, cell culture grade</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:152px;">146463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DPBS</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fetal bovine serum&#160;</td>
			<td style="width:191px;">Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MEM</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Bioconcept</td>
			<td>4-01F00-H&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X RIPA buffer</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:152px;">ab156034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SigmaFast protease inhibitor tablets</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">S8820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, sterile, flat-bottom, tissue culture treated Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, non-sterile, V-bottom Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-404</td>
			<td>used in the centrifugation step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">96-well plate, conical bottom, Nunc</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">249944</td>
			<td>compatible with the Acquity UHPLC system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">T75&#160; cell culture flasks Corning</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">CLS430641&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Peelable heat sealing foil&#160;</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;">186002789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acquity UPLC system</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Eppendorf Centrifuge 5810 R</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">05-413-323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Integration software: MassLynx V4.1</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Combi thermo-sealer SP-0669/240</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Quattro micro API Tandem Quadrupole System</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms

創薬における関心は高くなり新しいヒストン脱アセチル化酵素 (HDAC) 阻害剤の検索では。アイソ フォーム選択性ロミデプシン、クラス成立以来脚光を浴びている私は HDAC 阻害剤癌治療の多発性骨髄腫の特定の HDAC6 阻害剤の臨床的検討。現在のメソッドを使用して、HDAC1 とセルに HDAC6 試験化合物の阻害活性を決定します。アイソ フォームの活動は、超高速液体クロマトグラフィー-質量分析法 (クロマト ・ MS) 解析処理と未処理の HeLa 細胞と培養の特定の基板を使用して測定されます。メソッドには、分離のアイソ フォームで実施無料の細胞生化学的アッセイとは対照的、細胞環境で内因性 HDAC 活動を反映しての利点があります。さらに、それは合成基板の定量化に基づくが、ためメソッドは内生アセチル化タンパク質の抗体認識を必要ありません。それはいくつかの細胞株および自動化されたプロセスに容易に適応です。メソッドはすでに神経 HDAC6 選択的化合物を見つけることに有用な証明されています。典型的な結果をここに示します標準 HDAC 阻害剤トリコスタチン、(非特定の)、MS275 (HDAC1 固有の)、tubastatin (HDAC6 固有) を用いた HeLa 細胞と。

メソッドの HDAC1 の非選択性選択的阻害物質の識別への応用を実証する (クラス I)、HDAC6 (IIb クラス)、HeLa 細胞は知られている標準的な化合物と扱われた: トリコスタチン A (非選択的 HDAC1 と HDAC6)18MS275 (HDAC1 選択的阻害剤)19、および tubastatin (HDAC6 選択的阻害剤)20。本法 (図 1) を使用して、IC5...

Watch this Scientific Journal Video about Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS at JoVE.com

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

本手法は複数基板の UHPLC MS 解析によるヒストン脱アセチル化酵素 (HDAC) HeLa 細胞でのアイソ フォーム特異阻害剤を識別するために提供しています。これは生活 HDAC1 と HDAC6 の活動を反映するように開発された抗体無料メソッド セル環境、単一アイソ フォーム細胞アッセイとは対照的。

HDAC1 と HDAC6 hela 細胞活性の同時測定セルを用いたクロマト MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

simultaneous-measurement-hdac1-hdac6-activity-hela-cells-using-uhplc

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.4K ビュー.  Universités des Geneva–Lausanne (EPGL). 本手法は複数基板の UHPLC MS 解析によるヒストン脱アセチル化酵素 (HDAC) HeLa 細胞でのアイソ フォーム特異阻害剤を識別するために提供しています。これは生活 HDAC1 と HDAC6 の活動を反映するように開発された抗体無料メソッド セル環境、単一アイソ フォーム細胞アッセイとは対照的。

ビデオ: Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

ウルトラパフォーマンス液体クロマトグラフィー - 高分解能質量分析（UPLC-HRMS）を使用した生物源から非標的メタボロミクス

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

化学

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

DNAグリコシラーゼ活性の定常状態、プレ定常状態、およびシングルターンオーバー速度論的測定

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

蛍光を用いて藻体で中性脂肪を測定するための簡単​​かつ迅速なプロトコル

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

培養細胞および動物組織中のDNAの酸化バイオマーカー、8-オキソ-7,8-ジヒドロ-2&#39;-デオキシグアノシンのHPLC測定、

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

塩化物イオンの動的電気化学測定

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

酵素的、化学（メチル化）と物理（質量分析、核磁気共鳴）技術を用いた植物の壁ヘテロキシランのシーケンシング

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

光温度計の作製と試験

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

その力学的特性の測定を可能にする高強度共重合体のアラミド繊維を disentangling

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

単一金属ナノ粒子の高解像度物理特性評価

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction is to modulate the lipophilicity of the compound. In our group, we are interested in the study of the impact of fluorination on lipophilicity of aliphatic fluorohydrins and fluorinated carbohydrates. These are not UV-active, resulting in a challenging lipophilicity determination. Here, we present a straightforward method for the measurement of lipophilicity of fluorinated compounds by 19F NMR spectroscopy. This method requires no UV-activity. Accurate solute mass, solvent and aliquot volume are also not required to be measured. Using this method, we measured the lipophilicities of a large number of fluorinated alkanols and carbohydrates.

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

新しい簡単な19F NMR 分光学を使用して親油性 (logP) 測定法

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

HDAC1 と HDAC6 hela 細胞活性の同時測定セルを用いたクロマト MS

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この動画の章

ウルトラパフォーマンス液体クロマトグラフィー - 高分解能質量分析（UPLC-HRMS）を使用した生物源から非標的メタボロミクス

DNAグリコシラーゼ活性の定常状態、プレ定常状態、およびシングルターンオーバー速度論的測定

蛍光を用いて藻体で中性脂肪を測定するための簡単かつ迅速なプロトコル

培養細胞および動物組織中のDNAの酸化バイオマーカー、8-オキソ-7,8-ジヒドロ-2'-デオキシグアノシンのHPLC測定、

塩化物イオンの動的電気化学測定

酵素的、化学（メチル化）と物理（質量分析、核磁気共鳴）技術を用いた植物の壁ヘテロキシランのシーケンシング

光温度計の作製と試験

その力学的特性の測定を可能にする高強度共重合体のアラミド繊維を disentangling

単一金属ナノ粒子の高解像度物理特性評価

新しい簡単な¹⁹F NMR 分光学を使用して親油性 (logP) 測定法

HDAC1 と HDAC6 hela 細胞活性の同時測定セルを用いたクロマト MS

要約

さらに動画を探す

この動画の章

ウルトラパフォーマンス液体クロマトグラフィー - 高分解能質量分析（UPLC-HRMS）を使用した生物源から非標的メタボロミクス

DNAグリコシラーゼ活性の定常状態、プレ定常状態、およびシングルターンオーバー速度論的測定

蛍光を用いて藻体で中性脂肪を測定するための簡単​​かつ迅速なプロトコル

培養細胞および動物組織中のDNAの酸化バイオマーカー、8-オキソ-7,8-ジヒドロ-2'-デオキシグアノシンのHPLC測定、

塩化物イオンの動的電気化学測定

酵素的、化学（メチル化）と物理（質量分析、核磁気共鳴）技術を用いた植物の壁ヘテロキシランのシーケンシング

光温度計の作製と試験

その力学的特性の測定を可能にする高強度共重合体のアラミド繊維を disentangling

単一金属ナノ粒子の高解像度物理特性評価

新しい簡単な19F NMR 分光学を使用して親油性 (logP) 測定法

蛍光を用いて藻体で中性脂肪を測定するための簡単かつ迅速なプロトコル

新しい簡単な¹⁹F NMR 分光学を使用して親油性 (logP) 測定法