この作品は、KSSのNSF賞＃0920274およびサウスカロライナ州試験場プロジェクト（SC - 1700340）によってサポートされていました。本論文では、クレムソン大学の試験場の技術的貢献番号5929です。

このプロトコルの全体的なスキームを図1に概説されている。 1。標準曲線とアッセイのためのソリューションの準備<ol><li>ヒドロキシルアミン塩酸の2モル/ Lの溶液100 mLを準備します。ヒドロキシルアミン塩酸塩の13.9グラム（MW 69.49グラム/モル）を秤量し、約50 mLの蒸留-脱イオン水（のddH 2 O）に溶解する。水酸化カリウムペレットまたは濃縮液を用いてpHを7.0に調整する。 100mLのへの最終的なボリュームをもたらす。ソリューションは、最大30日間や4℃で90日まで室温で保存することができます。 </li><li>塩化第二鉄/塩酸溶液100mLを準備する。 13.5グラム塩化第二鉄（MW 270.32グラム/モル）を秤量し、約50 mLののddH 2 Oに溶解41.3 mLの濃塩酸（12.1モル/ L）を追加し、100mLの最終容量にもたらす。塩化第二鉄の最終濃度は0.5モル/ Lと塩酸の終濃度が5モルとなるようになります/ L.溶液を室温で保存されます。 </li><li> trichoroacetic酸1.84モル/ Lの溶液100 mLを準備します。トリクロロ酢酸（MW 163.39グラム/モル）の30.0 gを秤量、のddH 2 Oに溶解し、100mLの最終容量をもたらす。溶液を室温で保存されます。 </li><li> 0.1モル/ Lアセチルリン酸溶液5 mLを準備します。 0.092グラムアセチルリン酸（MW 184.06グラム/モル）を秤量、のddH 2 Oに溶解し、5 mLの最終的なボリュームをもたらす。注しマイクロ遠心チューブ（管当たり0.25 ml）に解決策と、使用時まで-20℃で凍結。解凍した溶液を氷に配置する必要がありますし、約1時間使用することができます。融解されたアセチルリン酸溶液は、再凍結になると、再使用しないでください。 </li><li> ADPの0.1mol / Lの溶液5mlを準備します。 ADP物0.24g（MW 472.5グラム/モル）を秤量、のddH 2 Oに溶解し、5 mLの最終的なボリュームをもたらす。 -20分注しマイクロ遠心チューブ内の溶液（試験管当たり0.5mL）を、凍結° Cまでの使用してください。使用するまで氷上で解凍したソリューションを保管してください。 </li><li>トリスの1モル/ Lの溶液50mlを準備します。 6.06グラムトリス（MW 121.14グラム/モル）を秤量し、40mLののddH 2 Oに溶解塩酸を用いて7.0に溶液のpHを調整し、50 mLの最終的なボリュームをもたらす。室温で保管ソリューション。 </li><li> 1モル/ L塩化マグネシウム溶液10 mLを準備します。 2.03グラムの塩化マグネシウム（MW 203.31グラム/モル）を秤量、のddH 2 Oに溶解し、10 mLまで最終的なボリュームをもたらす。室温でソリューションを保存してください。 </li><li>塩化第二鉄/塩酸溶液及びトリクロロ酢酸溶液の等量を混合して25 mLの開発ソリューションを準備。開発ソリューションは、毎日新しく調製し、室温で保存してください。 </li></ol> 2。アセチルリン酸の標準曲線の作成<ol><li>アセチルリン酸の標準曲線は、アセチルリン酸の既知量を使用して生成されます。 eighに6つの標準曲線tの点が適しています。規格は、0.1〜3ミリモル/ Lの最終濃度に相関する、0.03μモル〜300μLあたり0.9μモルまでの範囲とする300μLの最終容量で100mMトリス溶液中での適切な濃度にアセチルリン酸ストック溶液を希釈する。アセチルリン酸を含まない対照サンプルも準備する必要があります。 </li><li> ℃のヒートブロックで1分間37℃でそれぞれ標準とコントロールをインキュベートする。 </li><li>各規格へのヒドロキシルアミン塩酸塩溶液を3回振って、コントロールとミックス50μLを加える。 60℃のヒートブロックに標準を置き、5分間インキュベートする。 </li><li> 、規格及び制御するための開発溶液100μLを追加して三回振って混合し、色は、室温で少なくとも1分間のために開発することができます。 </li><li> 1分間18,000 × gで遠心機ですべての基準やコントロールを遠心分離します。 </li><li>分光光度計でサンプルを測定分光光度計用のブランクのようなコントロールを使用して、1mLのプラスチックキュベットを用いたT 540nmの。 </li><li>適切なデータ分析とグラフ作成ソフト（例：カレイダ、シナジーソフトウェア）を使用して標準曲線を生成します。 0.99以上のR 2値が望ましいです。データは、アセチルリン酸の存在のμモルに対し、540nmの吸光度としてプロットされます。 </li></ol> 3。酢酸キナーゼ活性のアッセイ<ol><li>説明するソリューションを使用して、0.1mol / Lのトリス緩衝液、10ミリモル/ L塩化マグネシウム、5ミリモル/ L ADP、および2ミリモル/ Lアセチルリン酸の最終濃度を含む反応ミックスを準備する。 10mLの反応混合物は8.2 mLののddH 2 O、1.0 mLのトリス溶液、0.1 mLの塩化マグネシウム溶液、0.5mLのADPのソリューション、および0.2 mLのアセチルリン酸溶液が必要になります。マイクロ遠心チューブに300μlのアリコートで反応ミックスを配布する。動力学的パラメーターと基質濃度の最適化の判定のために、一方の基板にすることができます変化や反応に一定の濃度で開催された他の。 </li><li> 37℃で1分間反応をプレインキュベート℃のヒートブロックインチ</li><li>反応する酵素の所望の量を加え、直ちに37℃のヒートブロックにチューブを返します。また、酵素なしが追加されていないため、コントロールの反応が含まれています。いくつかの反応が行われている場合、特定の時間間隔でチューブに酵素を加える。時間を追跡するようにしてください。 （注：使用する酵素の量は、アセチルリン酸またはADPの基板のすべてが枯渇されないことを保証するために最適化する必要があります - ディスカッションのセクションを参照してください） </li><li> 5分後、各反応と制御、ミックスにヒドロキシルアミン塩酸塩溶液の50μlずつを追加し、60℃のヒートブロックにチューブを置きます。いくつかの反応が実行されている場合、酵素が追加されたとして、これは同じ時間間隔で行われる必要があります。 </li><li> 60℃5分間反応とコントロールをインキュベート℃に</li><li> 100＆を追加します。ムー、各反応の開発ソリューションと制御、混合、およびラボのベンチ上にチューブのラック内の場所のL。色は、少なくとも1分間開発することができます。 </li><li> 1分間18,000 × gで遠心ですべてのチューブを遠心してください。 </li><li> 1mLのプラスチックキュベットを用いて540nmで分光光度計でサンプルを測定し、アセチルリン酸標準曲線と比較してアセチルリン酸の存在量を決定する。 </li><li>枯渇アセチルリン酸基質の量は、コントロールが欠け酵素に存在するアセチルリン酸の量から酵素反応後に残存するアセチルリン酸の量を差し引くことによって決定することができます。適切なデータとグラフ作成ソフト（例：カレイダ、シナジーソフトウェア）を使用してデータ分析を実行します。 0.97よりも大きいのR 2値が望ましいです。 </li></ol> 4。代表的な結果： この検定の目的は、直接的に酢酸キナーゼの活性を測定することです。酢酸の形成のイオン。酢酸の生産のための簡単​​な、直接的な測定法がないため、これは、アセチルリン酸基質の消費量を測定することによって行われます。酵素反応の間に所定の時間後に残っているアセチルリン酸はアセチルヒドロキへと続いて540nmで測定可能な赤みを帯びた色（図2）を持つ第二鉄ヒドロキコンプレックス、に変換されます。標準のμモルアセチルリン酸塩の存在対吸光度をプロットする標準曲線は、それぞれの反応に残っているアセチルリン酸の量を決定するために使用されます。標準曲線は、ゼロ点を通る直線になります。図3に示されている代表的な標準曲線は、データだけでなく、線形方程式をフィットを示し、モルアセチルリン酸の存在と0.99のR 2値あたり1.2332吸光度単位の傾きを持っています。検量線のデータに線形近似の式は、反応に残っているアセチルリン酸の量を計算するために使用されます。 μモル消費アセチルリン酸の酵素と酵素反応後に残存アセチルリン酸塩のμモルを欠く対照反応中に存在するアセチルリン酸塩のμモルの間の差として決定されます。 精製、組換えM.テルモの酢酸キナーゼは、記述されたプロトコルを用いて測定した。図4に示す実験では、ADPの濃度が5ミリモル/ Lで一定に保持し、反応のアセチルリン酸塩の濃度は、アセチルリン酸に対するK m を決定するために変化させた。予想通り、この実験では酵素は、それぞれの基質に対する標準的なミカエリス - メンテンのような動態を、次のとアセチルリン酸塩の濃度に対して活性をプロットするときに双曲線の飽和曲線を生成したことを示している。アセチルリン酸の見かけのK m値は0.27 ± 0.47の公開K m値に遜色0.01ミリモル/ L、±結合などを使用して0.01ミリモル/ Lであった13と言う。 直接アッセイはまた、標準的な結合アッセイを使用して不可能であるATP、以外の基質を利用する酢酸キナーゼの活性を測定するために使用することができます。組換え大腸菌を精製酢酸を形成する方向にI PPではなく、ATPを生成する赤痢アセテートキナーゼ16は 、ADPの代わりにリン酸ナトリウムを使用して、このアッセイに供した。反応でリン酸ナトリウムの濃度は0.2モル/ Lで一定に保持され、アセチルリン酸塩濃度を変化させた。 M.用としてテルモの酢酸キナーゼ、E.赤痢の酢酸キナーゼは、アセチルリン酸と双曲線の飽和曲線のミカエリス-メンテンのような動力学を追った（図5）が観察された。アセチルリン酸の見かけのK m値は0.50 ± 0.006ミリモル/ Lであった。リーブスとガスリー15はネイティブ酵素のアセチルリン酸0.06ミリモル/ LのK m値を決定し、どのように<html>これまで、それらの酵素は、部分的にしか精製され、それらのアッセイはまた、部分的にしか精製された4つのカップリング酵素を関与していた。したがって、これらの値を直接比較することは困難です。 プロトコルスキーム<ol><li>アセチルリン酸の標準曲線を生成する</li><li>所望の濃度に酵素を希釈する</li><li>マイクロ遠心チューブに反応混合物、アリコートを300μlを準備</li><li> 1分間で37 Preinc​​ubate反応が° C </li><li>特定の時間間隔で酵素を加えることによって反応を開始します。 </li><li>反応を停止し、60℃でインキュベートすることと同じ間隔でヒドロキシルアミンを追加℃で5分間</li><li>塩化第二鉄/カラーの開発のためのトリクロロ酢酸溶液を加える</li><li> 1分間18,000 × gでチューブを遠心</li><li> 540nmで吸光度を測定</li><li>標準曲線との比較で消費されるアセチルリン酸の量を決定する</li></ol>tp_upload/3474/3474fig1.jpg&quot;/&gt; 図1プロトコルスキーム <img alt="図2" src="/files/ftp_upload/3474/3474fig2.jpg" /> 図2。アセチルリン酸塩の規格 。アセチルリン酸塩濃度の増加は、ヒドロキシルアミンと色と反応させた説明したように開発されました。アセチルリン酸を欠くコントロールは明るい黄色で、アセチルリン酸の増加量を含有する反応物は色が連続的に暗いです。この色の変化は、540nmで測定されます。 <img alt="図3" src="/files/ftp_upload/3474/3474fig3.jpg" /> 図3サンプルアセチルリン酸の標準曲線。アセチルリン酸の様々な量は、ヒドロキシルアミンと反応させると発色液と吸光度を540nmで測定した。 μモルアセチルリン酸に対する540nmでの吸光度をプロットし、データへの線形近似を適用した。&lt;/ P&gt; <img alt="図4" src="/files/ftp_upload/3474/3474fig4.jpg" /> 図4。ATP産M.のアセチルリン酸の活用テルモの酢酸キナーゼ 。酵素活性は、アセチルリン酸の様々な濃度の5ミリモル/ L ADP存在下での酢酸の形成の方向で決定されたものです。アセチルリン酸の消費量は、反応や酵素反応後に残存アセチルリン酸の量のアセチルリン酸の開始量との差として決定された。アッセイは、示されたエラーバー付きの三連で行った。 <img alt="図5" src="/files/ftp_upload/3474/3474fig5.jpg" /> 図5。PP I産E.によるアセチルリン酸の活用赤痢の酢酸キナーゼ 。酢酸の形成の方向で酵素活性が0の存在下で決定された。アセチルリン酸の濃度を変えると、2モル/ Lリン酸ナトリウム。アセチルリン酸の消費量は、反応や酵素反応後に残存アセチルリン酸の量のアセチルリン酸の開始量との差として決定された。アッセイは、示されたエラーバー付きの三連で行った。

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+and+structural+characterization+of+the+novel+PPi-dependent+acetate+kinase+from+the+parasite+Entamoeba+histolytica.">Kinetic and structural characterization of the novel PPi-dependent acetate kinase from the parasite Entamoeba histolytica.</a> , (2011).</li><li>Lipmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+synthesis+of+acetyl+phosphate.">Enzymatic synthesis of acetyl phosphate.</a> J. Biol. Chem. 155, 55-70 (1944).</li><li>Lipmann, F., Tuttle, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+micromethod+for+determination+of+acyl+phosphates.">A specific micromethod for determination of acyl phosphates.</a> J. Biol. Chem. 159, 21-28 (1945).</li><li>Lipmann, F., Jones, M. E., Black, S., Flynn, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+ATP-CoA-acetate+reaction.">The mechanism of the ATP-CoA-acetate reaction.</a> J. Cell. Physiol. Suppl. 41, 109-112 (1953).</li><li>Rose, I. A., Grunberg-Manago, M., Korey, S. F., Ochoa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+phosphorylation+of+acetate.">Enzymatic phosphorylation of acetate.</a> J. Biol. Chem. 211, 737-756 (1954).</li><li>Allen, S. H., Kellermeyer, R. W., Stjernholm, R. L., Wood, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+enzymes+involved+in+the+proponic+acid+fermentation.">Purification and properties of enzymes involved in the proponic acid fermentation.</a> Journal of Bacteriology. 87, 171-187 (1964).</li><li>Clarke, P. M., Payton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+assay+for+acetate+in+spent+bacterial+culture+supernatants.">An enzymatic assay for acetate in spent bacterial culture supernatants.</a> Anal. Biochem. 130, 402-405 (1983).</li><li>Mukhopadhyay, S., Hasson, M. S., Sanders, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+assay+of+acetate+kinase+activity:+measurement+of+inorganic+phosphate+release+generated+by+hydroxylaminolysis+of+acetyl+phosphate.">A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.</a> Bioorg. Chem. 36, 65-69 (2008).</li><li>Nakajima, H., Suzuki, K., Imahori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+acetate+kinase+from+Bacillus+stearothermophilus.">Purification and properties of acetate kinase from Bacillus stearothermophilus.</a> J. Biochem. 84, 193-203 (1978).</li></ol>

利害の衝突は宣言されません。

このアッセイにおけるアセチルリン酸の検出は、ヒドロキシルアミン塩酸塩と塩化第二鉄溶液の濃度と酸性度の十分な濃度に依存している。アッセイ体積の変化は、これらのコンポーネントの両方の見直しが必要になります。酵素反応は、ここで説明し、37℃60℃実行ヒドロキシル終端を持つC℃で5分間で行った。これより高い温度は、アセチルヒドロキに残っているアセチルリン酸塩の迅速?...

<table border="1"><tr><td width="160"> 試薬の名前 </td><td width="108"> 会社 </td><td width="134"> カタログ番号 </td><td width="103"> コメント </td></tr><tr><td>アセチルリン酸</td><td>シグマアルドリッチ</td><td> 01409 </td><td>リチウム塩（97％） </td></tr><tr><td>ナトリウム一塩基性リン酸（脱水） </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td>ナトリウム二塩基性リン酸（無水） </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td>塩化マグネシウム（六水和物） </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td>トリスベース</td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td>塩化第二鉄（六水和物） </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td>トリクロロ酢酸</td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td>ヒドロキシルアミン塩酸塩</td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> 5&#39; -二リン酸アデノシンナトリウム塩</td><td>シグマアルドリッチ</td><td> A2754 </td><td></td></tr><tr><td> Biomate III分光光度計</td><td> ThermoFisher </td><td> 142982082 </td><td>標準的なUV /可視分光光度計</td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

酢酸キナーゼ、酢酸、砂糖キナーゼ- Hsp70の-アクチン（ASKHA）酵素のスーパーファミリー1-5のメンバーは、基質としてATPを利用したアセチルリン酸への酢酸の可逆的リン酸化を担当しています。酢酸キナーゼは、 古細菌の一属で見つかった、 細菌のユビキタスであり、またEukarya 6の微生物中に存在する。最もよく特徴付け酢酸キナーゼは、メタンガスを作り出す細菌Methanosarcinaサーモ 7月14日からということです。唯一のリン酸塩形成アセチル方向にATPない私を PPに利用できますが、酢酸キナーゼは、 赤痢アメーバ 、アメーバ赤痢の病原体から分離されており、これまでこの属15,16でのみ発見されました。 アセチルリン酸形成の方向では、酢酸キナーゼ活性は、通常、最初リップマンによって記述された、ヒドロキアッセイを用いて測定され17-20、ADPへのATPの変換が酵素のピルビン酸キナーゼ、乳酸脱水素酵素21,22、またはアセチルリン酸塩の製品との反応後の無機リン酸の放出を測定するアッセイによりNAD +にNADHの酸化に結合される結合アッセイヒドロキシルアミン23と。反対の活動は、酢酸を形成する方向は、ADPからNADPの還元+酵素のヘキソキナーゼとグルコース-6 -リン酸脱水素酵素24によりNADPHにするためにATPの形成を結合することによって測定されます。 ここでは、カップリング酵素を必要としない酢酸の形成の方向に酢酸キナーゼ活性を検出するための方法を説明しますが、代わりにアセチルリン酸の消費量の直接測定に基づいています。酵素反応の後、残りのアセチルリン酸塩は、ヒドロキアッセイ用として、分光光度法で測定することができる第二鉄ヒドロキ複合体に変換されます。このように、STとは異なり、ADPからATPの生産に依存しているこの方向のandard結合アッセイは、この直接的なアッセイは、ATPまたはPP iを生成する酢酸キナーゼのために使用することができます。

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

酢酸キナーゼ活性の測定方法について説明する。このアッセイは異なるホスホリルアクセプターで、酢酸を形成する方向に酢酸キナーゼの酵素活性と反応速度を決定するための直接的な反応を利用している。さらに、この方法は、酵素を利用する他のアセチルリン酸塩またはアセチル- CoAを検定するために利用することができます。

酵素酢酸キナーゼのアセテート形成活性の直接検出

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

direct-detection-acetate-forming-activity-enzyme-acetate

Research

JoVE Journal

Biology

23.0K ビュー.  Clemson University. 酢酸キナーゼ活性の測定方法について説明する。このアッセイは異なるホスホリルアクセプターで、酢酸を形成する方向に酢酸キナーゼの酵素活性と反応速度を決定するための直接的な反応を利用している。さらに、この方法は、酵素を利用する他のアセチルリン酸塩またはアセチル- CoAを検定するために利用することができます。

ビデオ: Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

生物学

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 ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME（免疫磁気電気化学的にリンクされた酵素）マイコトキシンの検出方法

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

トランスジェニックの生産アフリカツメガエル制限酵素を介した統合と核移植によって

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

一酸化窒素とスピントラップを使用して、細胞からの電子常磁性共鳴分光法によるスーパーオキシドアニオンラジカルの検出

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

FRETはレシオメトリックで細胞周期のキナーゼおよびホスファターゼの活動を監視

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2のキナーゼ活性を測定する in vitroで</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

土壌中の微生物の残留決定のためのAldononitrile酢酸誘導体化グルコサミンとムラミン酸のGCベースの検出

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活性の測定<em&gt;ショウジョウバエ</em&gt;アクティビティモニタ

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

成虫の蚊における排泄系の神経ペプチドおよび他の調節因子の活性の研究

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...