Work in our laboratory is funded by a VIDI grant from the Netherlands Organisation for Scientific research (to DJS). 
We thank Marc Stuart and the Department of Electron Microscopy at our university, for assistance with and providing access to the transmission electron microscope.

タグなしのFtsZタンパク質は、先に説明したように11,15 20mMトリス/塩酸（pH 7.9）、50mMのKCl、1mMのEGTA、2.5mMのMgAc及び10％グリセロールに対して透析し、精製し、そして-80℃で保存した。これらの条件下でタンパク質活性の有意な損失なしに2年以上保存することができる。タンパク質は解凍し、サンプルの凍結を回避するために、100μlアリコートに格納する必要があります。解凍後、サンプルは最大一週間4℃で保持することができる。 FtsZは、高濃度で可溶性であり、7〜10 mg / mlので保存することができる。これは、後の実験における貯蔵緩衝成分の望ましくない影響を回避するために、高タンパク質濃度を維持することが重要である。そこで、ストレージ濃度は少なくとも10倍、1％以下のグリセロール濃度をもたらすために、反応混合物中の透析緩衝液の他の成分の濃度を最小にするためののFtsZの希釈を可能にするはずである。 FtsZの重合はまた、高ナトリウム9の存在によって影響され得る</suP&gt;またはイミダゾールの濃度は、このように、これらのコンポーネントは、重合バッファから削除する必要があります。 7記載の-80℃での溶出バッファに格納されているようにSepFを精製したEからのFtsZの精製の ​​ための代替公表された手順大腸菌とB枯草菌ならびに異なる供給源からのFtsZの精製の ​​ための手順への参照は、（代表的な結果のセクションを参照） を表1にまとめる。 1。試料調製<ol><li>重合バッファを準備します。 <table border="1"><tbody><tr><td> 1 </td><td> 50mMのHepes / NaOHでpHを7.5 </td></tr><tr><td> 2 </td><td> 25 mMのパイプ/ NaOHでpHを6.8 </td></tr><tr><td> 3 </td><td> 50 mMのMES / NaOHでpHを6.5 </td></tr><tr><td> 4 </td><td> 50mMのHepes / NaOHを、pHが7.5、50mMのKCl </td></tr><tr><td> 5 </td><td> 25 mMのパイプ/ NaOHでpHを6.8、50のKCl </td></tr><tr><td> 6 </td><td> 50 mMの男ES / NaOHでpHを6.5、50のKCl </td></tr><tr><td> 7 </td><td> 50mMのHepes / NaOHを、pHが7.5、300 mMのKClを</td></tr><tr><td> 8 </td><td> 25 mMのパイプ/ NaOHでpHを6.8; 300のKCl </td></tr><tr><td> 9 </td><td> 50 mMのMES / NaOHでpHを6.5; 300のKCl </td></tr></tbody></table></li><li>フィルター22ミクロンのメンブランフィルターを用いてすべてのバッファを殺菌。 </li><li> （1）からの各重合液中100mMのGTP、GDPとのMgCl 2ストック溶液を準備します。 </li><li>全ての実験のために4℃で20分間、100,000×gでスピンによるタンパク質プリ·クリアー。 </li></ol> 2。 FtsZは沈降アッセイ<ol><li>高速遠心分離と互換性のあるチューブで（試料調製ポイント1を参照）の重合バッファの1にMgCl 2およびFtsZはを加えることにより反応ミックスを調製する。総体積は50μlの最終容量で、12μMで10 mmとMgCl 2およびFtsZのと、49μLにする必要があります。 </li><li> P、関数を振盪インキュベーター中でチューブレース300rpmで30℃で2分間インキュベートする（あるいは試料を穏やかに弾き飛ばされてもよいし、短時間、30℃で予熱に続いて微量遠心）。 </li><li> GTPまたは実験（最終濃度2mM）で用いたのと同じ緩衝液中のGDPのストック溶液1μlを添加することにより重合を開始。 300回転（または関数を振とう培養器内）で30℃、（それぞれ、50のKClおよび300mMのKClをバッファーなど）10、または2分間インキュベートします。 </li><li>超遠心ローターにチューブを移し、25℃で35万XG（TLA 120.1ローター用89700 rpm）で10分間スピンダウン</li><li>紡糸後、慎重にロータからチューブを取り外し、すぐにきれいなチューブに上清を移す。 2Xサンプルバッファー20μlに20μlの上清を添加することにより、SDS-PAGE用サンプルを準備します。 98℃で10分間沸騰させる</li><li>フォートを含むペレット画分に2×試料緩衝液50μlを追加SZポリマー。 2ミリリットルチューブに超遠心管を配置します。 98℃で10分間煮沸してペレットを再懸濁し、サンプル上清サンプルと同じ2倍希釈するために、脱イオンH 2 Oを50μlを加える。 </li><li> 2ミリリットルチューブに逆さまに超遠心管を回して、18,000 X gでエッペンドルフ遠心機で5分間スピンダウン。 2ミリリットル管から超遠心管を取り外します。 </li><li>負荷10％SDS-PAGEゲル上で並べて各上清およびペレット試料側を10μl。 150 Vでゲルを実行</li><li>クマシーブリリアントブルーG-250でゲルを染色し、定量化のために保管してください。 </li></ol> 3。スロースピンでのFtsZ沈降アッセイ<ol><li>ステップ2のように重合溶液を準備し、SepFおよび/またはFtsZは（最終濃度12μM）を追加します。これらの実験は、TLA-55ローターと組み合わせてベックマン使い捨て1.5mlチューブ中で行うことができる。 </li><li> FUを振とうしながら培養器にチューブをnctionおよび300rpmで30℃で2分間インキュベートする。 </li><li>ストック溶液（最終濃度2 mM）のからのGTPまたはGDPの追加1μLにより重合を開始し、300 rpmで30℃で20分間インキュベートする。 </li><li> 25℃で24,600×gで（TLA 55ローター、20,000 rpm）で15分間スピンダウン</li><li>上清を除去し、清潔なチューブに移す、20μLを取り、2×試料緩衝液20μlに移す。 98℃で10分間沸騰させる</li><li>脱イオンH 2 O50μlのを追加し、98℃で10分間煮沸することによりポリマーおよび再懸濁を含む画分をペレット化し、2Xサンプルバッファー50μLを追加</li><li>負荷10％SDS-PAGEゲル上で並べて各上清およびペレット試料側を10μl。 150 Vでゲルを実行</li><li>クマシーブリリアントブルーG-250でゲルを染色し、定量化のために保管してください。 </li></ol> 4。 FtsZは沈降アッセイの定量化<ol><li>クマシーSTAの写真を撮るゲルドキュメンテーションシステム（ 例えば 、LAS-4000、富士フイルム）を使用して、INEDゲル。 </li><li>ゲルのデンシトメトリー分析するのに適したプログラム（ 例えば AIDA画像解析プログラム（Raytest））でゲル画像を開いて、以下のプログラムで分析を説明します。 </li><li>ツールバーの「評価」ボタンをクリックしてください。 </li><li>リストから「2Dデンシトメトリー」を選択し、[OK]をクリックします。 </li><li>メインメニューで、「評価」をクリックして、「領域判定」エントリを選択します。 </li><li>ゲル上の領域を作成するには、領域判定ツールボックスの自動輪郭ツールのシンボルをクリックしてください。 </li><li> 、ゲル上のタンパク質バンドの左上隅をクリックし、マウスボタンを押したまま、バンドの所望の右下端にドラッグし、マウスボタンを離します。 </li><li>マークと同じ方法で、各のFtsZ / SepFバンド。 </li><li>割り当てられた領域の評価に関するすべての情報は、領域レポートに記載されています。レジオを入手するにはNレポートは、領域判定ツールボックスの「表示領域レポート」ボタンをクリックしてください。 </li><li>レポートをエクスポートするには、メインメニューの[ファイル]をクリックし、エントリ「2D領域レポートàエクスポート」を選択します。 </li><li>地域レポートでは、すべての作成された領域の強度が見つかるはず。レポートからの強度値を用いて、ペレット中のタンパク質のパーセント（FtsZの、調節タンパク質）を算出する。 </li></ol> 5。 90°光散乱<ol><li>蛍光分光計（ 例えば AMINCO-ボーマンシリーズ2）の電源を入れ、ランプは30℃でのキュベット室温度を維持するために熱変動を回避し、循環水浴をオンにするのに数分間ウォームアップすることができます</li><li>分光器の動作パラメータを定義します。検出器の高電圧300 V、発光および励起波長：350 nmのスリット幅：4 nmの。多くの分光器が自動的にcertAに実験開始時に信号を調整することに注意してください最大の割合（ 例えば 60％）である。これは、すぐに、GTPがFtsZのを重合するために追加されているように範囲外に移動するための散乱信号を発生します。これは、最初の一連の実験を開始する前に、右信号増幅検出器の高電圧に対する最大の重合を確立することが賢明です。 </li><li>データ収集プロトコルをプログラムします。 3600秒の持続時間、時間ベースの取得を選択します。 </li><li> 5分間、室温の水浴に必要 - 超音波処理した場合、水とエタノールとの蛍光キュベットを丁寧に清掃してください。このプロトコルでは、1cmの経路長及び200μlの容積を有するキュベットが使用され、貯蔵溶液（0.5〜1％のHellmanex）に格納される。代替として、単回使用の適合性プラスチックUVキュベット（UVette、エッペンドルフ）を使用することができる。 </li><li>最終的な300μLに対して計算濃度、およびVORTとして10 MgCl 2および12μMのFtsZはと（サンプル調製ポイント1を参照）の重合バッファのマスターミックスの294μLを準備EX。 </li><li>キュベットに重合バッファー196μLを移し、分光器に入れてください。 30℃で2分間インキュベートする。 </li><li>データ収集を開始し、信号が安定していることを確認するために90秒間待ちます。 </li><li> 100のGTPまたはGDP（最終濃度：2 mm）で4μl加え200μlの最終反応容量を達成するために、買収をミックスして再開するより大きな容量ピペットで上下にピペット。 </li></ol> 6。透過型電子顕微鏡<ol><li> （ボリュームは、材料を節約するために、10μlの最終容量を削減することができる）のFtsZ沈降アッセイ用としてサンプルを準備します。 </li><li>インキュベーション時間の後、グロー放電さ400メッシュの炭素被覆銅グリッド上に試料を2μLを適用し、15秒間インキュベートする。 </li><li>優しくグリッド並列または濾紙に垂直に触れたりドラッグ​​して、グリッド乾燥ブロット。 </li><li> 2％の酢酸ウラニル溶液を4μLを使用してグリッドを染色。 BLOステップ6.3で説明したように、グリッドのドライT。 </li><li> 39200倍の倍率で120 kVので動作するフィリップスCM120電子顕微鏡グリッドを表示します。 </li></ol> 7。 GTP加水分解アッセイ実験の設定は、FtsZのGTPの加水分解はマラカイトグリーンとの反応物を混合することにより、種々の反応時間後に停止されるように設計されている。このようにして、マラカイトグリーンの発色の時間は、各試料について同じである。 <ol><li>重合バッファの1つに1.5ミリリットル2のGTPを準備します（サンプル調製、ポイント1を参照）。 重合液中の0-40μMリン酸標準希釈液を調製し、POMG-25H（バイオアッセイ）キットで説明したようにマラカイトグリーンの作業試薬を調製。あるいは、作業試薬はLanzetta らによって記載されたプロトコルに従って社内で調製することができる。16 </li><li>総量360μlのマスターミックスを準備します。 <table国境= &quot;1&quot;&gt; <tbody><tr><td>私</td><td> 24μMのFtsZ BS、20のMgCl 2、重合液</td></tr><tr><td> 2 </td><td> 12μMのFtsZ EC、20のMgCl 2、重合液</td></tr><tr><td> III（コントロール1） </td><td> 24μMのFtsZ BS、2mMのEDTA、重合液</td></tr><tr><td> IV（コントロール2） </td><td> 12μMのFtsZ EC、2mMのEDTA、重合液</td></tr></tbody></table></li><li>ピペットマスターミックス、100μlのリン酸基準や定量PCR 96ウェルプレート中2のGTP180μlを20μlのアリコートを次の順序で。 <table border="1"><tbody><tr><td></td><td> A </td><td> B </td><td> C言語</td><td> D </td><td> E </td><td> F </td><td> G </td><td> H </td></tr><tr><td> 1 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </TD&gt; <td> 2 </td><td> 2 </td></tr><tr><td> 2 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 3 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 4 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 5 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 6 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 7 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td> &lt;/ TR&gt; <tr><td> 8 </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 9 </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td></tr><tr><td> 10 </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td></tr><tr><td> 11 </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 12 </td><td> GTP </td><td> GTP </td><td> GTP </td><td> GTP </td><td> GTP </td><td> GTP </td><td> GTP </td><td> GTP </td></tr></tbody></table> PS - リン酸標準</li><li> 30℃で40分間周期に設定されたプログラムでPCR機で定量PCRプレートを置き</li><li> malac20μlのアリコートを準備96ウェルプレート中のハイト緑色作業試薬。 1&#39;-8 &#39;レーンの実験から、重合液の60μlを加える。サンプルは、リン酸標準と比較4Xに希釈され、最終的な計算中に、この事実を考慮してください。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> A </td><td> B </td><td> C言語</td><td> D </td><td> E </td><td> F </td><td> G </td><td> H </td></tr><tr><td> 1 &#39; </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 2 &#39; </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 3 &#39; </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 4 &#39; </td> &lt;TD&gt; III </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 5 &#39; </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 6 &#39; </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 7 &#39; </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 8 &#39; </td><td> 3 </td><td> 3 </td><td>私</td><td>私</td><td> 4 </td><td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> 9 &#39; </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td></tr><tr><td> 10 &#39; </td><td> PS </td&gt; <td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td><td> PS </td></tr></tbody></table></li><li> （これは30分の反応の開始時点で）、1レーンへのGTPの20μl加えピペットを上下に混合する、タイマーを開始します。 </li><li> 10分後、30秒、2レーンへのGTPを追加ピペットを上下（20分反応）を混合する。 </li><li> 16分後に上下3、ピペット混合する（15分反応）レーンにGTPを追加します。 </li><li> 21分後、30秒（10分反応）をミックスするには、上下4、ピペットをレーンにGTPを追加します。 </li><li> 25分後（7分反応）混合する、ピペットを上下5レーンへのGTPを追加します。 </li><li> 27分後、30秒（5分反応）をミックスするには、上下6、ピペットをレーンにGTPを追加します。 </li><li> 29分後（4分の反応）を混合する、ピペットを上下7レーンへのGTPを追加.. </li><li>レーン1にレーン1から30分の転送を20μlの後で、上下のピペット混合する（3マラカイトグリーンの発色の出発点0分反応）。 </li><li> 30分後、30〜2レーン2から秒の転送20μL &#39;、ピペットを上下（20分の反応のためにマラカイトグリーンの発色の開始点）を混合する。 </li><li>レーン3-7ごとに30秒間のステップを繰り返します。 </li><li> 33分後、30秒のために（マラカイトグリーンの発色のための0分の反応と開始時点を混合するピペットを上下、8レーンへのGTP20μlのピペットを上に追加して、ミックスダウンすると「8レーンに20μLを転送反応）。 </li><li> 34分後、「レーン9〜9を上下にピペット混合する（リン酸標準1用のマラカイトグリーンの発色の開始時点）を80μLを加える。 </li><li> 34分30秒の転送80μlの後には、上下車線から10から10 &#39;、ピペット混合する（リン酸標準2用のマラカイトグリーンの発色の開始時点）。 </li><li>サンプルから全ての気泡を除去し、さらに25分間室温でプレートをインキュベート30秒。 </li><li> 96ウェルプレートリーダーでプレートを置き。 </li><li> 60分後（マラカイトグリーンの発色は、最初の反応のために今30分である）、波長630 nmでプレート10倍ごとに30秒を測定します。すべての測定は、ステップ7.6から7.12と7.16からのすべての時点に対応し、データ解析の際に別々に計算しなければなりません。 </li><li>リン酸標準較正曲線を用いて各サンプル中の遊離リン酸を計算する。 </li><li>時間をかけてリン酸の放出などのデータをプロットし、曲線の直線範囲からのFtsZのGTP加水分解活性を計算します。 </li></ol>

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No conflicts of interest declared.

We describe a set of methods that allows a quick analysis of FtsZ activity and its interaction with other proteins. Light scattering, sedimentation and GTPase assays as well as electron microscopy have been widely used to study FtsZ polymerization. We have made some improvements to existing protocols, we showed the influence of different conditions on FtsZ assembly, and we propose controls that should be included in FtsZ studies.
We introduce low speed centrifugation to distinguish large structures formed by the association between FtsZ and its interacting proteins from FtsZ polymers. This method shows two advantages over the standard sedimentation assay. First, no background is formed by the FtsZ polymers in the pellet fraction as they are not spun down at 24,600 x g. Second, the amount of FtsZ present in the structure formed with an interacting protein may be calculated from the gel. Two critical steps in this method are the incubation time and the GTP concentration. It is important to centrifuge the large protein structure when it is complete but before it disassembles when all GTP is hydrolyzed. The best control for this study is polymerization of FtsZ with GDP. There is one potential limitation of the assay. FtsZ forms a stable complex with SepF, which can easily be spun down at 24,600 x g. If the sedimentation with another activator or a drug that bundles FtsZ polymers is performed, it may be necessary to adapt the assay. It may be done by changing the incubation time, or increasing the speed of centrifugation.
Proper preparation of the sample is the most important for light scattering experiments. Proteins must be precleared by spinning and all the buffers should be filtered prior to use. If any aggregates are present in the sample, they will disrupt a stable signal obtained from FtsZ polymers. For the analysis of the FtsZ structures by electron microscopy, preparation of a grid is the main step. The time of sample incubation on the grid will have the effect of producing more or less compacted polymers. For bundles of FtsZBs, the time of incubation must be shorter than for FtsZEc and FtsZBs at high KCl concentration. We used a concentration of 12 &#181;M for every sample to be able to compare the results. However, for FtsZBs at 50 mM KCl a lower FtsZ concentration should be used, as 12 &#181;M resulted in a full saturation of the grid. This makes the polymers highly compacted and difficult to detect. Less compacted polymers are better to detect on EM.
The GTPase assay is the only experiment used to study the activity rather than the structures of FtsZ. Mg2+ is necessary for GTP turnover in FtsZ polymers. Thus, in the absence of Mg2+, FtsZ does not hydrolyze GTP. Therefore, a sample with no Mg2+ is the right control in this assay but cations of Mg are present in the FtsZ storage buffer. They may be removed by addition of 1 mM EDTA to the control sample. The critical step in this assay is the incubation time. It is important to stop FtsZ activity after a given time. This is achieved by transferring the FtsZ sample to a malachite green solution in a 96-well plate. However, development of the malachite green color is a continuous process. Thus the measurements must be taken at the same time for every sample. Using a well-planned GTP addition protocol with measurements taken each 30 sec apart in an established order, it is possible to obtain the same incubation and sample handling time for every time point. Another critical step is choosing the concentration of the protein for the experiment. In the experiment we used two different concentrations for FtsZEc and FtsZBs. GTP hydrolysis is much quicker for FtsZEc compared to FtsZBs. The GTPase activity of FtsZEc under chosen conditions and at 12 &#181;M is linear only for maximum&#160;5 min and after that time the hydrolysis rate plateaus. Thus, it is difficult to interpret data from the experiment when performed under these conditions. In this case FtsZEc must be used at lower concentration than FtsZBs to be able to compare activities of both proteins. The GTPase activity of FtsZs from different sources may vary. Thus, the right concentration must be chosen. The concentration for FtsZ polymerization should be well above the critical concentration (in general from 2.5-10 &#181;M). The dynamics of FtsZ assembly and disassembly is also important. Some proteins show a significant lag in polymerization after addition of GTP, as shown for FtsZBs at 50 mM KCl. It is useful to perform the light scattering assay before the GTPase assay to approximate the time of assembly and disassembly of FtsZ polymers. After that, the time of incubation and concentration of protein may be chosen. Since the conditions chosen for FtsZ polymerization are crucial, it is important to use the right pH and KCl concentration in each method. In this work we studied 9 different buffers with pH ranging from 6.5-7.5 and KCl concentrations from 0 M to 300 mM. We noticed that the best condition to analyze FtsZs from B. subtilis and E.coli and their biological activity is at pH that is close to physiological level (7.5) together with a high KCl concentration. At a high KCl concentration, FtsZ has a higher GTPase activity and produces polymers that are better detectable by electron microscopy. We also confirmed that the physiological pH and a high KCl concentration are better for the study of the interaction between FtsZ and regulatory proteins than any other buffers mostly used to study FtsZ assembly. FtsZBs shows a similar activity to FtsZEc when studied at high KCl concentration. In addition, at low salt concentration the influence of pH is more visible than in the buffers with high salt concentration. FtsZ sometimes precipitates when using buffers without KCl, as a result, buffers without salt should be avoided. Sedimentation of FtsZ polymers is low when using buffers with high KCl concentrations. This may be an advantage when studying interactions between FtsZ and proteins that assemble FtsZ filaments such as SepF and ZapA as these higher order structures are easy to detect with centrifugation. In all our experiments we used MgCl2 at a 10 mM concentration. It was shown that a relatively high Mg2+ concentration stabilizes FtsZ polymers and reduces the GTPase activity of FtsZ. In Table 2 results from various studies are summarized describing FtsZ polymerization and GTPase activity at different Mg2+ concentrations using otherwise identical buffer conditions 27. The measured concentration of free cytoplasmic Mg2+ is 0.9 mM3. It should be noticed that GTP will chelate an equivalent amount of Mg2+. Thus, the optimal Mg2+ concentration for GTPase experiments is around 2-2.5 mM, which is close to physiological level3. However, in our experiments we used MgCl2 at a 10 mM concentration to obtain an easily detectable light scattering signal and to stabilize FtsZ polymers during the sedimentation assay.
Although we applied our protocols to FtsZ from the model organisms E. coli and B. subtilis, they can be adapted to FtsZ from any other organism. It has to be noted that the physiological pH, and concentrations of monovalent, and divalent cations differ among organisms. Thus, the optimal conditions for FtsZ polymerization may vary. Differences in doubling time and growth conditions of different bacteria may result in different assembly kinetics of FtsZ and optimal conditions of the experiments. However, our protocol provides a good starting point for the experiments with FtsZs from other organisms. The protocols should be useful for the study of FtsZ with regulatory proteins or the study of effects of small compounds and drugs on FtsZ.
<table border="1">
	<tbody>
		<tr>
			<td>Source</td>
			<td>Polymerization [% of FtsZ sedimented]</td>
			<td>GTPase [Pi/FtsZ/min]</td>
			<td>Mg2+ concentration [mM]</td>
			<td>FtsZ concentration [&#181;M]</td>
			<td>References</td>
		</tr>
		<tr>
			<td>FtsZEc</td>
			<td>~ 28%</td>
			<td>~ 2.1</td>
			<td>10</td>
			<td>12</td>
			<td>This work</td>
		</tr>
		<tr>
			<td></td>
			<td>~ 50%</td>
			<td>~ 2.4</td>
			<td>10</td>
			<td>12.5</td>
			<td>27</td>
		</tr>
		<tr>
			<td></td>
			<td>~ 43%</td>
			<td>~ 3.5</td>
			<td>5</td>
			<td>12.5</td>
			<td>27</td>
		</tr>
		<tr>
			<td></td>
			<td>~ 27%</td>
			<td>~ 4.6</td>
			<td>2.5</td>
			<td>12.5</td>
			<td>27</td>
		</tr>
		<tr>
			<td></td>
			<td>ND</td>
			<td>~ 5.4</td>
			<td>2.5</td>
			<td>5</td>
			<td>26</td>
		</tr>
		<tr>
			<td>FtsZBs</td>
			<td>~ 30%</td>
			<td>~ 0.8</td>
			<td></td>
			<td>12</td>
			<td>This work</td>
		</tr>
		<tr>
			<td></td>
			<td>~ 52% (with DEAE dextran)</td>
			<td>~ 0.5</td>
			<td>10</td>
			<td>10</td>
			<td>11</td>
		</tr>
		<tr>
			<td></td>
			<td>ND</td>
			<td>~ 2.25</td>
			<td>2.5</td>
			<td>5</td>
			<td>26</td>
		</tr>
	</tbody>
</table>
Table 2. Effect on Mg2+ on FtsZ polymerization and GTPase. Results from this work compared to published data. All experiments were carried out in 50 mM MES/ NaOH, pH=6.5, 50 mM KCl.

必須の細菌タンパク質FtsZは、細菌の細胞分裂機構の最もよく特徴付けられたタンパク質である。 FtsZは、チューブリンの原核生物相同体であるとGTP依存的にin vitroで重合する。 FtsZは、細菌の1,2への保存された自然や独自性のために新しい抗生物質のために非常に魅力的な標的である。細胞分裂の開始時に、FtsZは、他の細胞分裂タンパク質の組み立てのための足場として働くmidcellにおける細胞質分裂の環を形成する。 Z-環の形成は、分割面の正しい位置特定のために重要である。のFtsZの組立ダイナミクスは、MINC、SepF、ZapA、UgtP、エズラ2（細菌種に応じて）など、いくつかのアクセサリータンパク質によって調節されています。 FtsZの重合は、インビトロで集中的に研究されており、直プロトフィラメント、湾曲したプロトフィラメント、フィラメントのシート、フィラメントのフィラメント、チューブの束を含む多くの異なる構造がデスされている組立バッファ、ヌクレオチド、およびアッセイ3に含まれている追加のタンパク質に依存しcribed。 カウロバクターのクレセンタスにおける電子cryotomography実験は、Zリングが豊富な4をバンドルすることなく、比較的短い、非連続シングルプロトフィラメントから組み立てられていることを示唆しているが、生体内でのFtsZのプロトフィラメントのアーキテクチャは、まだ完全には理解されていません。 in vitroでは 、FtsZの重合特性及び調節タンパク質とのFtsZの相互作用は、反応バッファーの組成に敏感である。例えば、我々は最近、FtsZのC末端にSepFための相互作用部位を説明し、FtsZは宿泊 Δ16C末端TRUNCATEはもはやSepF 5に結合することが示されなかった。 SepF-FtsZは宿泊の相互作用に関するこれまでの研究では、同様のFtsZ 宿泊 Δ16はまだSepFがFtsZの上のセカンダリサイトに結合することが示唆されSepFとcosedimentedとtruncate <suP&gt; 6。これらの研究の違いは、pH7.5でバッファ - のFtsZとSepFのない共沈降は、pH6.5の共沈降があったのに対し、切り捨てるありませんでした。反応の組成物であった。 Gündoğdu ら 。 SepFが機能していないし、pH 6.5で観察共沈降がSepFの析出はなく、FtsZの宿泊 Δ16C末端TRUNCATEとの相互作用によって引き起こされる可能性が高いことを示し、pH6.5の7に析出と指摘した。のFtsZの重合に対するpHおよびKCl濃度の影響は、以前に検討されている。 EのポリマーpH6.5で大腸菌のFtsZ（FtsZはEC）は 、より長く、より豊富な中性pH 8,9で形成されたものよりも。 Tadros ら 。バインディングのK +が FtsZのEcの重合にリンクされ、FtsZは活動10のために重要であることは注目に一価の陽イオンの存在下でのFtsZ のEcの重合を検討した。 pHがよりcriticaですSepFの前の例、およびFtsZは11日 MINCの抑制効果のpH依存性で示すように、他のタンパク質とのFtsZの相互作用は、検討されているL。 pHおよび塩濃度の両方が他のタンパク質とのFtsZの相互作用に影響を及ぼし得るように、FtsZの重合試験のための適切な条件およびコントロールを選択することが重要である。 ここでは、光散乱、電子顕微鏡、沈降、およびGTPアーゼアッセイによりFtsZの重合およびGTPase活性を研究するためのプロトコルを記述します。直角光散乱は、即座に12 FtsZの重合を研究するための標準的な方法である。私たちは、沈降およびGTPアーゼアッセイ、数の改善を導入しました。我々は、光散乱および電子顕微鏡用の試料を調製する方法を詳細に述べる。 FtsZの重合を研究する文献で使用されるいくつかのバッファがテストされ、我々は、各実験のための最高の条件を説明した。我々はまたあるべきかを制御示して最適なデータを得るために導入した。 これらのメソッドは、ほとんどの実験室で提供され、簡単な方法および装置を使用して他のタンパク質とのFtsZの重合活性および相互作用の迅速な研究を可能にする。 FtsZの重合を研究するより洗練された方法が存在するが、多くの場合、蛍光標識8,13,14とのより専門的な機器へのアクセス、および/ ​​またはFtsZのの修正が必要。このホワイトペーパーで説明の簡単な方法は、BからのFtsZを使用して説明している枯草菌および大腸菌大腸菌 、最も一般的なグラム陽性およびグラムモデル生物。プロトコルは、他のFtsZタンパク質に適合させることができる。これらの新規FtsZsによる予備アッセイに基づいて、インキュベーション時間、緩衝液または温度に関するわずかな変化は、最適な結果のために必要であり得る。ここに記載された実験は、これらの最適な条件を見つけるのに役立つはずです。

<table border="0" cellpadding="0" cellspacing="0" width="1048">
	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">GTP</td>
			<td style="width:381px;">Roche</td>
			<td align="right" style="width:119px;">10106399001</td>
			<td style="width:167px;">Part 1, 2, 3, 4, 5, 6, 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thickwall Polycarbonate Tubes</td>
			<td style="width:381px;">Beckman Coulter</td>
			<td align="right">343776</td>
			<td>Part 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Optima MAX-XP Ultracentrifuge</td>
			<td style="width:381px;">Beckman Coulter</td>
			<td align="right">393315</td>
			<td>Part 2, 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyallomer Tube with Snap-on Cap</td>
			<td style="width:381px;">Beckman Coulter</td>
			<td align="right">357448</td>
			<td>Part 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">AIDA Bio-package, 1D, 2D, FL</td>
			<td style="width:381px;">Raytest Isotopenmessger&#228;te GmbH</td>
			<td align="right" style="width:119px;">15000001</td>
			<td>Part 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Luminescence Image Analyzer LAS-4000</td>
			<td style="width:381px;">Fujifilm</td>
			<td style="width:119px;"></td>
			<td>Part 4</td>
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			<td height="42" style="height:42px;width:381px;">Thermo Spectronic AMINCO-Bowman Luminescence Spectrometer</td>
			<td style="width:381px;">Spectronic Instruments</td>
			<td style="width:119px;"></td>
			<td>Part 5</td>
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			<td height="21" style="height:21px;width:381px;">Fluorescence Cell</td>
			<td>Hellma Analytics</td>
			<td>105-250-15-40</td>
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			<td height="21" style="height:21px;">Square 400 Mesh, Copper, 100/vial</td>
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			<td>Part 6</td>
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			<td height="21" style="height:21px;">CM120 Electron Microscope Operating at 120 kV</td>
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			<td>Part 6</td>
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			<td height="21" style="height:21px;">96 ml x 0.2 ml Plate</td>
			<td style="width:381px;">BIOplastics</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Malachite Green Phosphate Assay Kit</td>
			<td style="width:381px;">BioAssay System</td>
			<td style="width:119px;">POMG-25H</td>
			<td>Part 7</td>
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			<td height="21" style="height:21px;">PowerWave HT Microplate Spectrophotometer</td>
			<td style="width:381px;">BioTek</td>
			<td style="width:119px;"></td>
			<td>Part 7</td>
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</table>

ftsz polymerization assays: simple protocols and considerations

細菌の細胞分裂の間、必須タンパク質のFtsZは、いわゆるZ-環を形成して、セルの中央に組み立てる。 FtsZは、 インビトロで GTPの存在下で長いフィラメントに重合し、重合は、いくつかのアクセサリータンパク質により調節される。 FtsZの重合は、広範囲に光散乱、沈降、GTP加水分解アッセイおよび電子顕微鏡法などの基本的な方法を用いてインビトロで研究されてきた。バッファー条件の影響は、両方のFtsZの重合特性、および調節タンパク質と相互作用するためのFtsZの能力。ここでは、FtsZの重合研究のためのプロトコルを記述し、モデルタンパク質として大腸菌、枯草菌のFtsZを使用して条件とコントロールを検証します。低速沈降アッセイは、バンドルまたはFtsZの重合体をチューブ状のタンパク質とのFtsZの相互作用の研究を可能に導入される。改良されたGTPアーゼアッセイプロトコルは、テストを可能に記載されているリン酸検出反応での発色の変化を廃止し、標準化インキュベーション時間を有する96ウェルプレートセットアップにおいて、種々の条件を用いて経時的にGTP加水分解。光散乱試験および電子顕微鏡観察用試料の調製が記載されている。複数のバッファは、FtsZの重合の研究に適した緩衝液pHおよび塩濃度を確立するために使用される。塩化カリウム高濃度の実験のほとんどのための最善の方法です。我々の方法は、E.からだけでなく、のFtsZ のインビトロ特徴付けのための出発点を提供大腸菌とB枯草菌が、他の細菌から。このように、本方法は、調節タンパク質またはFtsZの重合に影響を与える可能性抗菌薬のテストとのFtsZの相互作用の研究のために使用することができる。

Purification of FtsZ from different bacterial sources has been described in the literature and is summarized in Table 1.
<table border="1">
	<tbody>
		<tr>
			<td>Source</td>
			<td>Method</td>
			<td>Modification</td>
			<td>Yield obtained [mg/L of culture]&#62;</td>
			<td>References</td>
		</tr>
		<tr>
			<td>B. subtilis</td>
			<td>1) Ammonium sulfate precipitation/ion exchange chromatography</td>
			<td>no</td>
			<td>40</td>
			<td>This work, 11,15</td>
		</tr>
		<tr>
			<td>2) Affinity chromatography</td>
			<td>His-tag</td>
			<td>ND</td>
			<td>17</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli</td>
			<td>1) Ammonium sulfate precipitation/ion exchange chromatography</td>
			<td>no</td>
			<td>35</td>
			<td>This work, 11,15</td>
		</tr>
		<tr>
			<td>2) Calcium precipitation, ion exchange chromatography</td>
			<td>no</td>
			<td>40</td>
			<td>18</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanococcus jannaschii</td>
			<td>1) Affinity chromatography under denaturing conditions/refolding/ammonium sulfate precipitation/gel filtration</td>
			<td>His-tag</td>
			<td>1.3</td>
			<td>19</td>
		</tr>
		<tr>
			<td>2) Affinity chromatography/ gel filtration</td>
			<td>His-tag</td>
			<td>ND</td>
			<td>20</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermotoga maritima</td>
			<td>Ion exchange chromatography/gel filtration</td>
			<td>no</td>
			<td>6.7</td>
			<td>19</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa</td>
			<td>Affinity chromatography/gel filtration</td>
			<td>Strep-tag, His-tag</td>
			<td>ND</td>
			<td>21</td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis</td>
			<td>1) Affinity/ion exchange chromatography</td>
			<td>no</td>
			<td>ND</td>
			<td>22</td>
		</tr>
		<tr>
			<td>2) Affinity chromatography/ gel filtration</td>
			<td>no</td>
			<td>30</td>
			<td>23</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquifex aeolicus</td>
			<td>Affinity chromatography/gel filtration</td>
			<td>His-tag, C-terminal truncation (331-367)</td>
			<td>ND</td>
			<td>24</td>
		</tr>
		<tr>
			<td>Caulobacter crescentus</td>
			<td>Ion exchange chromatography/ammonium sulfate precipitation/gel filtration</td>
			<td>no</td>
			<td>ND</td>
			<td>25</td>
		</tr>
	</tbody>
</table>
Table 1. FtsZ purification protocols described. ND: not determined.
Sedimentation of FtsZ polymers
Initially, we used two different velocities to spin down FtsZ polymers. We found that only at a velocity of 350,000 x g single polymers of FtsZEc are spun down (Figure&#160;1) whereas at 190,000 x g only bundles of FtsZBs are present in the pellet fraction (data not shown). Therefore 350,000 x g was used in our further experiments. The percentage of polymerized FtsZEc and FtsZBs is similar at 50 mM KCl even though the light scattering experiments revealed a much higher scattering signal for FtsZBs. This is due to bundles formed by FtsZBs which scatter more light than single polymers of FtsZEc. It was not possible to obtain high amount of FtsZ polymers in the pellet fraction in the experiment with 300 mM KCl for both FtsZEc and FtsZBs (Figure&#160;1). We attribute this to a combination of quick disassembly of the FtsZ structures and decreased bundling of the filaments.
Sedimentation of FtsZ-SepF tubules
To analyze the interaction of FtsZ with certain activators sedimentation assays can be performed at lower centrifugation speeds. At this velocity only large structures of FtsZ may be pelleted, e.g.&#160;the large tubules formed by SepF rings and FtsZBs filaments5, or the bundles formed by FtsZ and ZapA. We used lower centrifugation (24,600 x g) to demonstrate the feasibility of this approach for the tubules formed by FtsZ and SepF. FtsZ was recovered in the pellet above background levels only when both SepF and GTP were present in the sample (Figure&#160;2), and the presence of SepF does not influence FtsZ GTPase activity7 showing that FtsZ is fully active in the presence of SepF. Specific sedimentation of SepF and FtsZ is roughly 45% of total SepF and 15% of total FtsZ (compared to material sedimenting when GDP is added). This shows that the SepF-FtsZ tubules contain more SepF than FtsZ. This may be because many SepF rings organize the FtsZ-SepF tubules5,7. The exact stoichiometry of SepF-FtsZ in these tubules is not known but our results suggest that there is more SepF present than FtsZ.
FtsZEc and FtsZBs polymerization and bundling properties
To characterize the polymerization efficiency of FtsZBs and FtsZEc in different buffers we analyzed both proteins by 90&#176; angle light scattering. At 50 mM KCl, FtsZBs gives a 20-40-fold higher light scattering signal than FtsZEc depending on buffer pH (Figures&#160;3A and&#160;B) confirming results of Buske et al.26&#160;Increasing the KCl concentration in the buffer did not significantly influence the light scattering signal of FtsZEc (Figure&#160;3D) but the signal of FtsZBs decreased ~80-fold at pH 7.5, ~30-fold at pH 6.8 and ~45-fold at pH 6.5 in 300 mM KCl (Figure&#160;3C) compared to buffers with 50 mM KCl (Figure&#160;3A). Disassembly of FtsZ polymers is faster at higher KCl concentration for both proteins (Figures&#160;3C and&#160;D). Studies of Pacheco-G&#243;mez et al. show that E. coli FtsZ polymerization and bundling is pH dependent. These authors found that in a buffer with 50 mM KCl the light scattering signal of FtsZ polymerization was higher, and disassembly of FtsZ took longer at pH 6.0 compared to pH 7.0 8. These results are not in agreement with our data from polymerization of FtsZEc at 50 mM KCl (Figure&#160;3B), but it has to be noted that we have used three different buffers (HEPES, MES and PIPES) where Pacheco-G&#243;mez et al. only used MES. Thus, not only the pH, but also buffer composition (ionic strength) affects the kinetics of FtsZ polymers at 50 mM KCl. However, at 300 mM KCl neither buffer composition nor pH influenced FtsZEc assembly in a detectable manner.
A light scattering experiment of FtsZBs in buffers without KCl was not possible due to precipitation of the protein under these conditions. When the concentration of FtsZBs was lowered to 3 &#181;M, precipitation did not occur. However, 3 &#181;M is not the physiological concentration of FtsZ in the cell. In plastic cuvettes, FtsZBs did not precipitate at 12 &#181;M at pH 7.5, but at pH 6.8 and 6.5 FtsZ still precipitated in the absence of KCl.
Morphologies of FtsZ structures from E. coli and B. subtilis
The structures formed by FtsZ were inspected by TEM. FtsZBs assembled into closely compacted polymers that covered the whole grid in all buffers at low salt (Figures&#160;4A-C). FtsZEc formed long filaments, cables and bundles in all buffers at low salt (Figures&#160;4D-F). However, the observable amount of polymers formed by FtsZEc was lower than the amount formed by FtsZBs. In high salt buffers FtsZBs formed longer single-stranded protofilaments which did not associate into bundles (Figures&#160;4G-I). While FtsZBs protofilaments changed structure at higher salt concentration, FtsZEc formed structures indistinguishable from those of low salt buffer (Figures&#160;4J-L). There was no observable pH influence on polymerization of FtsZEc but FtsZBs forms more bundles at pH 6.5 which are visible as closely compacted polymers and sheets (Figure&#160;4B). These results are in accord with our light scattering experiments and previously published TEM work8,11,26.
The GTPase activity of FtsZ at high and low KCl concentrations
The GTP hydrolysis activity of FtsZ was measured under different conditions using a colorimetric assay for free phosphate. As reported previously15 the GTPase activity of FtsZ increased with increasing KCl concentration: depending on the buffer used FtsZBs. showed a 3-7 fold increase, and FtsZEc showed a 1.5-2.5 fold increase in GTPase activity at 300 mM KCl compared to 50 mM KCl. The reduced GTPase activity at 50 mM KCl is due to bundling of FtsZBs filaments. At 50 mM KCl FtsZEc had a 3-6 fold higher GTPase activity than FtsZBs due to quicker disassembly of the FtsZEc polymers. The difference in GTP hydrolysis activity between FtsZBs and FtsZEc was reduced at 300 mM KCl, possibly because of reduced bundling of FtsZBs filaments (Figure&#160;5).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50844/50844fig1.jpg" width="600px" /> 
Figure 1. Quantification of FtsZ polymerization by sedimentation.&#160;(A) 12 &#181;M FtsZ was polymerized in the presence of 2 mM GTP or GDP at pH 7.5 (black bars), 6.8 (grey bars) or 6.5 (white bars). The amount of protein pelleted was determined by densitometric analysis of Coomassie stained gels. GDP served as a control for a specific sedimentation and the percentage of FtsZ sedimented with GDP was subtracted from the percentage of FtsZ sedimented with GTP to obtain the values plotted in the graph. On the left: FtsZ sedimented at 50 mM KCl, on the right: FtsZ sedimented at 300 mM KCl. (B) Representative results from Coomassie stained gels. Polymerization of FtsZBs (upper gel) and FtsZEc (lower gel) at 50 mM KCl. (S) supernatant, (P) pellet fractions from the experiment. <a href="https://www.jove.com/files/ftp_upload/50844/50844fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50844/50844fig2.jpg" width="600px" /> 
Figure 2. Sedimentation of SepF/FtsZ tubules at low speed. (A) 12 &#181;M FtsZBs was polymerized with 2 mM GDP (white bars) or GTP (black bars). The amount of protein pelleted was determined by densitometric analysis of Coomassie stained gels. The + and - signs under the x-axis indicate the presence or absence of FtsZ and SepF in the reaction. (B) Representative results from a Coomassie stained gel. Polymerization of FtsZBs in the presence and absence of SepF. As a control SepF without FtsZ was used. Polymerization was carried out with GTP and GDP. (S) supernatant, (P) pellet fractions from the experiment. <a href="https://www.jove.com/files/ftp_upload/50844/50844fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50844/50844fig3.jpg" width="600px" /> 
Figure 3. Light scattering of 12 &#956;M FtsZEc and 12 &#956;M FtsZBs. FtsZs were assembled in the presence of 2 mM GTP and polymerization was monitored by 90&#176; angle light scattering. Polymerization of FtsZBs (A) and FtsZEc (B) at 50 mM KCl at pH 7.5, pH 6.8, pH 6.5. Polymerization of FtsZBs (C) and FtsZEc (D) at 300 mM KCl at pH 7.5, pH 6.8 and pH 6.5. <a href="https://www.jove.com/files/ftp_upload/50844/50844fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/50844/50844fig4.jpg" width="600px" /> 
Figure 4. Structures of FtsZBs and FtsZEc polymers visualized by electron microscopy. (A-L) Images of 12 &#181;M FtsZBs (A-C and G-I) and FtsZEc (D-F) and (J-L) polymerized with 2 mM GTP. (A-C) FtsZBs in buffer with 50 mM KCl and pH 7.5, 6.8, and 6.5 respectively. (D-F) FtsZEc in buffer with 50 mM KCl and pH 7.5, 6.8, and 6.5 respectively. (G-I) FtsZBs in buffer with 300 mM KCl and pH 7.5, 6.8, and 6.5 respectively. (J-L) FtsZEc in buffer with 300 mM KCl and pH 7.5, 6.8, and 6.5 respectively. Scale bar: 100 nm. <a href="https://www.jove.com/files/ftp_upload/50844/50844fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/50844/50844fig5.jpg" width="600px" /> 
Figure 5. GTP hydrolysis during FtsZ polymerization in 6 different buffers.&#160;In all experiments 2 mM GTP was used. As a control sample with no MgCl2 was used. Activity of FtsZ without MgCl2 was subtracted from the activity of FtsZ in the presence of MgCl2. On the left: GTPase activity of FtsZ at 50 mM KCl, on the right: GTPase activity of FtsZ at 300 mM KCl. <a href="https://www.jove.com/files/ftp_upload/50844/50844fig5highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about FtsZ Polymerization Assays: Simple Protocols and Considerations at JoVE.com

FtsZ Polymerization Assays: Simple Protocols and Considerations

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZは重合アッセイ：シンプルプロトコルと考慮事項

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

ftsz-polymerization-assays-simple-protocols-and-considerations

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JoVE Journal

Biology

15.1K ビュー.  University of Groningen. Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

ビデオ: FtsZ Polymerization Assays: Simple Protocols and Considerations

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

バキュロウイルスによるチョウ目幼虫の経口感染するためのプロトコル

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

生物学

Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be infected with microapplicator techniques in the gut with baculovirus polyhedra and in the hemolymph with budded virus. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents.  Formulation and application of baculoviruses for pest control purposes are described elsewhere.

In this video, we demonstrate two microapplicator techniques used to infect of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

バキュロウイルスによるチョウ目幼虫のMicroapplicatorアシスト感染するためのプロトコル

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

内タンパク質の定方向進化のための変異導入と機能選択プロトコル E.大腸菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

精巣細胞懸濁液の調製のためのシンプルで効率的な手法

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

インビトロでの細胞遊走および浸潤アッセイにおける

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

高リン酸化タウタンパク質を用いたインビトロ凝集アッセイにおいて

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

準備、イメージング、および細菌表面運動性アッセイの定量化

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

デジタル核酸定量アッセイの簡単な一括読み出し

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...