The authors thank Kerstin Glensk for preparing the MTases M.BseCI and Set7/9 and gratefully acknowledge funding by the Excellence Initiative of the German Federal and State Governments and RWTH Aachen University. The authors are happy to provide 6BAz and SeAdoYn or other cofactor analogues for collaborative research.

1.一般手順<ol><li>ストアアジリジン補助因子（DMSO中）6BAzとタンパク質MTアーゼ-80℃でSET7 / 9と二重活性化補助因子SeAdoYnとDNA MTアーゼM.BseCI -20℃（50％グリセロール）を含むすべての他の試薬。 </li><li>脱イオン水に吸光係数ε269nmで （6BAz）= 16000センチメートル-1 M -1およびε260nmの （SeAdoYn）= 15400センチメートル-1 M -1用いたUV /可視分光法を経由して 6BAzとSeAdoYnの濃度を決定する。吸光係数は、280nmでの直接吸収を介して利用可能である場合には、ブラッドフォードアッセイによってMTアーゼの濃度を決定するか。 </li><li>酵素活性の損失を防ぐために、集中的なペッティングまたはボルテックスにより気泡を作成しないようにしてください。その代わりに、優しく上下にピペッティングにより混合します。 </li><li> DMSO中のストック溶液からアジリジン補助因子を追加する場合はアッセイにおける最終DMSO濃度は低いことを確認してください5％未満。常にDNAとの非特異的反応を防止するアッセイ緩​​衝液中の10mMのマグネシウムイオンが挙げられる。 </li><li>酸性のストック溶液から二重に活性化補助因子を追加する場合はpH変化を回避し、アッセイ溶液のpHが大きく変化しないことを確認するために小容量（高濃縮ストック溶液）を使用します。彼らは必要な銅イオンの錯体形成によってクリック反応を妨害することができるので、アッセイ緩衝液中で、チオールを避けるため、 例えば 、βメルカプトエタノールまたはジチオスレイトール（DTT）。 </li></ol>アジリジン補因子を介した DNAの2つのステップのラベル<ol><li> M.BseCI DNA MTアーゼおよびアジリジン補助因子6BAzプラスミドDNAの配列特異的メチルトランスフェラーゼ誘発ラベルING（笑顔）。 <ol><li> 20℃における補因子ソリューションを解凍し、氷上で反応混合物を準備します。 </li><li>検定に加えて任意の非固有の変更を可視化するために、「補因子」の制御を行う、とAN＆＃8220。酵素」コントロールは、MTアーゼの準備は、自然補因子のAdoMetがないことを確認します。 </li><li> （100 mMトリス-塩酸を含む、100のMgCl 2、20 mMのβメルカプトエタノール、pH7.4）で10倍の修正バッファ、pBR322の2μL（0.5μgの/μL）、10当量のアッセイミックス2μlのために。 DNA上の認識配列（pBR322の中で1認識配列）と20μlの総体積内の60μMの最終濃度にアジリジン補助因子6BAzあたりM.BseCI。最後の補因子とDNA MTアーゼを追加します。 注：βメルカプトエタノールは、毒性、腐食性、環境に有害である。 </li><li> 「補因子」制御のために代わりにM.BseCIの脱イオン水を追加し、「酵素」コントロールには6BAzの代わりに脱イオン水を追加します。 </li><li>優しく上下にピペッティングすることによって溶液を混合。 </li><li> 1時間55℃でチューブをインキュベートします。 </li><li>チューブの底にすべての液体を回収するための遠心分離を簡単に。 </li></ol></li><li> DNA修飾を確認するための制限修飾アッセイ。 <ol><li> 、80μlの脱イオン水および3.3液（100mMのトリス-HCl、50mMのMgCl 2を 、1 MのNaCl、1mg / mlのウシ血清アルブミン、pHが8.0を含む）10μlの10×R.TaqI緩衝液を混合して溶液を調製サーマス·アクアティクス （R.TaqI、10 U /μL）から制限エンドヌクレアーゼ（REase）。最後のステップでREaseを追加してください。 </li><li>各チューブに2.1.7から10倍のR.のTaqIバッファと上から溶液（2.2.1）の28μLの2μlを添加する。 </li><li>優しく上下にピペッティングすることによって溶液を混合。 </li><li> 30分間65℃でチューブをインキュベートします。 </li><li>チューブの底にすべての液体を回収するための遠心分離を簡単に。 </li></ol></li><li>ストレプトアビジンとの電気泳動移動度シフトアッセイ（EMSA）は、機能的な変更を確認します。 <ol><li>各チューブ（2.2.5）からの25μlのを削除し、ストレプトアビジンMに対するストレプトアビジン溶液（1 mMと2.4μlを添加するonomerの100mMのNa 2 HPO 4を含有するストレプトアビジン緩衝液中で、100mMのNaCl、pH7.5で、総ビオチンの4当量）。残りのチューブにストレプトアビジンバッファーの2.4を添加する。 </li><li> 1時間、37℃ですべてのチューブをインキュベートします。 </li></ol></li><li>アガロースゲル電気泳動による分析。 <ol><li>各チューブに6Xローディングバッファー（0.25％ブロモフェノールブルー、30％グリセロール）の5μlを添加する。 </li><li>優しく溶液を混合。 </li><li>負荷（10,000倍ストック溶液から1×GelRedを含む0.5×TBE緩衝液中の1％アガロース）アガロースゲルのウェルに10μlの各サンプル。 </li><li>約80 Vで0.5×TBE緩衝液でゲルを実行します。 1時間。 </li><li>フィルタ（540±50 nm）とを備えたCCDカメラ（312 nm）のUVテーブル上でDNAバンドを可視化する。 注：UV光が目や皮膚に損傷を与えている。 </li></ol></li></ol>ダブル活性化補助因子を介した 3つの段階のタンパク質標識<ol><li> MethyltransfヒストンH3リジン4ラベリング（改質工程）のためのSET7 / 9と二重活性化補助因子SeAdoYnで活性化グループ（MTAG）の譲渡を消去し、指示した。 <ol><li>コンポーネントを解凍し、氷上で反応混合物を準備します。注：必ずSeAdoYn劣化を避けるために冷却して続ける。 </li><li>アッセイに加えて、任意の非特異的改変を可視化するために、「補因子」制御を実行し、「酵素」制御は、蛍光プローブの非特異反応を除外する。 </li><li>修正緩衝液（50mMのTris-HCl、5％グリセロール、pH8.5）中、10μMヒストンH3、10μMSET7 / 9及び600μMSeAdoYn（セレンの両エピマーの混合物）を含有するアッセイ溶液（20μl）を調製する。最後のステップでは、次に補因子とMTアーゼを追加します。 </li><li> 「補因子」コントロール3.1.3のようにアッセイ溶液を準備し、合成補助因子と競争する60 mMののAdoMetを追加します。 「酵素」制御のために代わりにSeAdoYnの脱イオン水を追加します。</li><li>ゆっくり上下にピペッティングすることによって溶液を混合。 pHのストリップ（ - 10 pH範囲5）の上部のフィールドに各溶液1μlを添加してpHを確認してください。 </li><li> 2時間37℃でインキュベートする。 </li><li> 1：0.1％（w / v）のAPS、0.04％の（v / v）のTEMED 12％アクリルアミド/ビスアクリルアミド37.5 357 mMのビス - トリスpHが6.5〜6.8、：一方のゲルを実行して（12％SDSポリアクリルアミドゲルを調製で;ローディングゲル：357 mMのビス - トリスpHが6.5〜6.8、0.1％APS（w / v）の、0.04％（v / v）のTEMEDと5％アクリルアミド/ビスアクリルアミド37.5：1）。 注：アクリルアミド/ビスアクリルアミドは毒性と健康に危険である。この手順の間に手袋を着用してください。 </li></ol></li><li>銅触媒アジド-アルキン環（CuAAC）（標識工程） を介してヒストンH3でalkinylatedリジン4の化学標識。 <ol><li>修飾反応の終了は、3 mMのCuSO 4を、3 mMのトリス（3-ヒドロキシプロピルトリアゾリル）アミン（THPTA）、250mMのアスコルビン酸ナトリウムおよび6 mMのTAMRAアジドを含有する5倍クリックミックスを調製する直前20μlの総容量。 </li><li> CuAACを開始し、修飾反応をクエンチするために各チューブに調製したばかりの5倍のクリックミックスの5μlを添加する。 </li><li>ピペッティングにより穏やかに混合します。 </li><li>フォアの光退色を避けるために、光からアルミホイルで全てのチューブを保護します。 </li><li> 1時間20℃でインキュベートする。 </li></ol></li><li>無料のTAMRAフルオロフォアの過剰を除去するタンパク質沈殿。 <ol><li> 34 -無料のTAMRAの蛍光団の集中的なゲル内蛍光によって蛍光ラベルヒストンH3のoutshiningを回避するために、タンパク質（3.3.4 3.3.2）の沈殿によって過剰蛍光団を取り除く。 </li><li>簡単に各添加後、各チューブとボルテックスに75μlのメタノール、18.8μlのクロロホルムと50μlの脱イオン水を追加します。 5分間16000×gで遠心分離する。タンパク質が含まれている界面層を、乱すことなく上相を除去します。 </li><li>私は残りの相に56.3μlのメタノールを追加します。nは5分間16000×gで各チューブ、ボルテックスと遠心機は、タンパク質をペレット化する。上清を取り除きます。ペレットを洗浄するには、この手順を繰り返します。 </li><li>糸くずの出ない組織とのオープンチューブをカバーし、15のためにそれらを乾燥させ - 30分。 </li></ol></li><li> SDS PAGE による分析。 <ol><li> （20μlのSDSローディング緩衝液（50mMトリス-HCl、2.5％（w / v）のSDS、10％（v / v）グリセロール、320 mMのβメルカプトエタノールおよび0.05％で3.3.4から沈殿したタンパク質を溶解/ワットv）のブロモフェノールブルー、pHは6.8）。完全にピペットでチューブの壁をすすぐことによりペレットを溶解することを確認してください。 </li><li> 10分間95℃でサンプルをインキュベートし、それらを20℃まで冷却しましょう​​。 </li><li>チューブの底にすべての液体を回収するための遠心分離を簡単に。 </li><li> SDSポリアクリルアミドゲル（3.1.7）のウェルに各サンプルの全量をロードします。電気泳動用のバッファを実行しているようにSDSを（w / v）の50 mMのMOPS、50mMのTris-X（Tris塩基）、5mMのEDTA、0.1％を使用してください。 </li><li>約120 Vでゲルを実行します。 90分。 </li><li>フィルタ（50ナノメートルの波長±540）を備えたCCDカメラを用いてUVテーブル（312 nm）の上のゲル内蛍光を可視化する。 注：UV光が目や皮膚に損傷を与えている。 </li></ol></li></ol>

The authors disclose the following competing financial interest: E.W. is inventor on related patents.

のDNA MTアーゼおよびアジリジン補助因子（笑顔DNA）を用いたDNAのワンステップ標識は堅牢な方法であるが、実験を計画する際にいくつかの側面を考慮すべきである。 アジリジン補助因子：M.BseCIとDNA標識用6BAz濃度は60であった。他のDNAメチルトランスフェラーゼを使用する場合は補因子の濃度は、20μMのがDNA MTアーゼM.TaqI 19で採用されていると低い例え?...

核酸、1,2およびタンパク質3,4の特異的な標識は、機能の特徴付け、医療診断及び（ナノ）バイオテクノロジーのための主要な関心事である。ここでは、S -adenosyl-L-メチオニン（AdoMetをまたはSAM）依存性メチルトランスフェラーゼ（MTアーゼ）に基づいており、これらの生体高分子のための酵素標識法を提示する。このクラスの酵素（EC 2.1.1。）は、核酸およびタンパク質の特定の残基の中の個々の求核位置（窒素、酸素、硫黄および炭素原子）を標的と自然補因子のAdoMet（ 図1A）5の活性化メチル基を転移する。また、MTアーゼは、親和性タグ、蛍光団又は他の標識（ 図1B）6との特異的標識のための合成補因子類縁体を利用することができます。のAdoMet類似体の2つのクラスが開発されてきた：Sの equence固有のM ethyltransferase-ためのアジリジン補助因子を私が </strong&gt; nduced Lのアベルる （笑顔）7とctivated のG roups のM ethyltransferase指向性T ransferのための二重活性化したのAdoMet類縁体（MTAG）8。 <img alt="図1" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig1highres.jpg" width="700" /> 図1：メチルトランスフェラーゼ（MTアーゼ）によって触媒される反応は 、DNA、RNA、タンパク質および小生体分子を含む様々な基材への天然の補因子のAdoMet（SAM）からA.メチル基転移B.標識核酸およびタンパク質の/官能化（NNNNN =。 DNAのための塩基対、タンパク質についてのRNAおよびアミノ酸に対するヌクレオチド;緑色対象残基とMTアーゼのXXXXX =認識配列）合成補因子類似体。レポーター基を含むアジリジン補助因子（青球）特に標的残基に結合された配列は、（左）アデニン環にある取り付け及び二活性化のAdoMet類似体は、第二段階でバイオ直交クリック反応により標識することができる化学レポーターY（右）を担持する拡張アルキル鎖の転送につながる。 <a href="https://www.jove.com/files/ftp_upload/52014/52014fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> アジリジン補助因子は、DNAメチルトランスフェラーゼで最適に動作する。それらは窒素原子9（またはNが 10,11 -mustard）の代わりに、スルホニウム中心の反応性基を有する3員環を含む。この窒素原子のプロトン化は、DNAと全体の補因子の共有結合につながる標的ヌクレオチドによる求核攻撃のためのアジリジン環を活性化させる。アデニン環にレポーター基を結合することにより、アジリジン補因子は、（一段階でDNAを標識するためにDNAメチルトランスフェラーゼと組み合わせて使用​​することができる<stron<html> G&gt;左図1B）7,12。 、 バチルス·ステアロサーモフィラス （M.BseCI）16（ 図2から図15（アデニン環の6位に結合したビオチンとアジリジン補助因子）およびアデニン特異的DNA MTアーゼ-これは6BAz 13によるDNAのビオチン化のために詳細に示されているアジリジン補助因子を介した DNAのワンステップのラベリング）：プロトコルセクション2を参照してください。 ヘモフィルスheamolyticus（M.HhaI、5からM.BseCI（5&#39;-ATCG A T-3 &#39;認識配列）、 サーマス·アクアティクス （M.TaqI、5&#39;-TCG A -3からDNA MTアーゼ&#39;）に加えて、 「-G C GC-3 &#39;）およびスピロ （M.SssI、5&#39;- C G-3&#39;）から正常に6BAz 17とのDNAをビオチン化するために使用されている。さらに、アジリジン補因子は、1ステップの蛍光DNA標識18,19のために使用することができる。 常に &quot;&gt;：&quot; =キープtogether.withinページFO」ontent <img alt="図2" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig2highres.jpg" width="700" /> 図2：M.BseCIと6BAzとDNAの配列特異的一工程ビオチン化 DNA MTアーゼM.BseCIは、二本鎖DNA配列5&#39;-ATCG A T-3 &#39;を認識し、自然に二アデニンのアミノ基をメチル化のAdoMetを使用して残留物（緑色）。アジリジン補助因子6BAzとの反応のコースが変更され、M.BseCIは、ターゲットアデニンとビオチン（青）を含めた全体の補因子を結合することによって、特定のDNAビオチン化を配列決定するためにつながる。 <a href="https://www.jove.com/files/ftp_upload/52014/52014fig2highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 二重活性化のAdoMet類似体は、不飽和側鎖の代わりに、スルホニウム中心におけるメチル基（ 図1B拡張含む</stronG&gt;、右）20。スルホニウム中心部へのβ位に不飽和二重または三重結合は、電子的に接合安定化により遷移状態の中不利な立体効果を補償します。スルホニウム中心と不飽和結合の両方が酵素転送のための側鎖を活性化するので、これらの補因子は、二重活性化のAdoMet類似体と命名した。典型的には、それらは、第二工程8,21において化学選択的標識のために、アミノ、アルキンとアジド基のようなユニークな化学基（化学レポーター）で側鎖を転送するために使用される。 RNAおよびタンパク質の追加のラベル付けを可能28 -一般的には、二重の活性化のAdoMet類似体は、RNA MTアーゼ22,23 DNA MTアーゼ8,20,21のためだけでなく、ための補因子およびタンパク質のMTアーゼ24としてだけではなく機能することができます。しかし、拡張された側鎖が立体的にメチル基より厳しいであり、タンパク質工学によってMTアーゼ活性部位を拡大することはしばしばある効率的な転送速度を得るために必要なEN。銅による化学的修飾を、以下のための酵素の転写時の遷移状態の1安定化および2反応性ハンドル：この問題に対する別の解決策は、小さなプロパルギル末端アルキンは、2つの機能を果たす基（3個の炭素）とのAdoMet類似体を使用することである触媒によるアジド - アルキン環（CuAAC）ケミストリーをクリックします。それは、結果のプロパルギルのAdoMet類縁体29は 、中性またはわずかに塩基性条件下でのみ限定された使用の非常に不安定であることが判明した。この欠点は、セレンと硫黄原子を置換することによって固定することができる。得られた補因子5 &#39; - [（ セレン ）[（3 S）-3-アミノ-3-カルボキシプロピル]プロプ-2- ynylselenonio] -5&#39;-デオキシアデノシン（SeAdoYn、 図3）は、野生型DNAによって受け入れられ、RNA多くの場合、タンパク質工学の必要性を排除32 -とタンパク質MTアーゼ30。これは、蛍光プロ例示される<html>ヒストンH3リジン4（H3K4）MTアーゼSET7 / 9 33（：二重に活性化補助因子を介して 2つの段階のタンパク質標識、図3、プロトコルセクション3を参照）での標識をTEIN。 <img alt="図3" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig3highres.jpg" width="700" /> 図3：SET7 / 9、SeAdoYnとTAMRAアジドとヒストンH3の配列特異的二段階蛍光標識タンパク質MTアーゼSET7 / 9が自然のAdoMetを使用して、ヒストンH3のリジン4のアミノ基（H3K4、緑）をメチル化する。。二重活性化補助因子でSeAdoYn MTアーゼは、リジン残基に小さなプロパルギル基（赤）を転送する。接続された端末三重結合は、その後選択的にアジド誘導体化TAMRA（テトラメチルローダミン、青）フルオロフォアでバイオ直交クリック反応（銅触媒アジド - アルキン付加環、CuAAC）に変更されます。ロード/ 52014 / 52014fig3highres.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name of Reagent/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">6BAz</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Weinhold et al., Patent number US 8,129,106, published March 6, 2012.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">&#946;-Mercaptoethanol</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">28625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acetic acid</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10304980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">10688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">UltraPure Agarose</td>
			<td style="width:139px;">Invitrogen</td>
			<td align="right" style="width:119px;">16500100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ammonium persulfate (APS)</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">13375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bis-Tris</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Boric acid</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bromophenol blue Na salt</td>
			<td style="width:139px;">Serva</td>
			<td align="right">15375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Copper(II) sulfate</td>
			<td style="width:139px;">Aldrich</td>
			<td style="width:119px;">C1297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Chloroform</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right">10020090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Coomassie Brilliant Blue</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">17525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EDTA disodium salt</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethanol</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">100983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">GelRed (10000x in water)</td>
			<td style="width:139px;">Biotium</td>
			<td align="right" style="width:119px;">41003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Glycerol (99.5%)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">FastRuler Low Range DNA Ladder</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SM1103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Histone H3</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Philipp Voigt and Prof. Danny Reinberg; expression and isolation according to T. J. Richmond et al., J. Mol. Biol. 1997, 272, 301-311.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M.BseCI</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Michael Kokkinidis; expression and isolation according to Kapetaniou et al., Acta Cryst. 2006, F63, 12-14.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Methanol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10675112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesiumchloride&#160; hexahydrate</td>
			<td style="width:139px;">J.T. Baker</td>
			<td align="right" style="width:119px;">4003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">MOPS</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium chloride</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pH strip (Neutralit)</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">1,095,330,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pBR322</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SD0041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">R.TaqI (10u/&#181;l)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>ER0671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SeAdoYn</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Willnow et al., ChemBioChem 2012, 13, 1167-1173.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Set7/9</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Prof. Danny Reinberg, expression and isolation according to D. Reinberg et al., Genes Dev.2002, 16, 479-489.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Streptavidin</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">3058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">(+)-Sodium L-ascorbate</td>
			<td style="width:139px;">Sigma Life Science</td>
			<td style="width:119px;">A7631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SDS Granular</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">di-Sodiumhydrogenphosphat</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">106,586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TAMRA-azide</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TaqI buffer (10x)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">B28</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:245px;">N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td style="width:139px;">Acros Organics</td>
			<td align="right" style="width:119px;">42058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-HCl</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-X (TRIS-base)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1018</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Tris(3-hydroxypropyltriazolyl-methyl)amine (THPTA)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">762342</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues

S -Adenosyl -1-メチオニンメチオニン（AdoMetまたはSAM）依存性メチルトランスフェラーゼ（MTアーゼ）、DNA、RNA、タンパク質および小生体分子の特定の位置にあるAdoMetから活性化メチル基の転移を触媒する。この天然のメチル化反応は、補因子の合成類似体を用いてアルキル化反応の多種多様に拡張することができる。アジリジン環とのAdoMetの反応性のスルホニウム中心の交換は、様々なDNAメチルトランスフェラーゼによって、DNAと結合することができる補因子につながる。これらのアジリジン補助因子は、アデニン部分の異なる位置にレポーター基を搭載し、 私は DNA（笑顔DNA） のLアベルのINGを nduced ethyltransferase- のS equence固有のMのために使用することができる。典型的な例として、我々は、DNA MTアーゼM.BseCIとアジリジン補助因子6BAzのある5&#39;-ATCG A T-3 &#39;配列でpBR322プラスミドDNAのビオチン化のためのプロトコルを与える1ステップ。 ctivated Gの roups（MTAG） のm個のethyltransferase指向Tの ransferのために使用されるのAdoMet類似体の別のクラスの不飽和アルキル基の結果と活性化されたメチル基の拡張。拡張された側鎖はスルホニウム中心と不飽和結合によって活性化されるので、これらの補因子は、二重活性化のAdoMet類似体と呼ばれる。これらの類似体アジリジン補因子のようなDNAメチルトランスフェラーゼのための補因子としての機能だけでなく、RNA、タンパク質および小分子MTアーゼのみならず。これらは、典型的には、第2の化学工程でレポーター基で標識されているユニークな官能基を有するMTアーゼ基質の酵素的修飾に使用される。これは、ヒストンH3タンパク質の蛍光標識のためのプロトコルに例示されている。小さなプロパルギル基はのクリックラベルに続いてヒストンH3リジン4（H3K4）MTアーゼSET7 / 9によってタンパク質に補因子アナログSeAdoYnから転送されるTAMRAアジドとアルキニル化ヒストンH3。補因子類縁体とMTアーゼ媒介ラベリングは、識別とMTアーゼ基質の機能的研究ならびにDNAジェノタイピングとメチル化検出を含む多くのエキサイティングなアプリケーションを可能にする技術である。

アジリジン補因子を介したDNAのワンステップラベリング この例では、反応は、二本鎖の5&#39;-ATCG A T-3 &#39;配列内の第2のアデニン残基を変更し、pBR322プラスミド（ 図4A）上の1つの認識部位を持つDNA MTアーゼM.BseCI、を用いて行われる。プラスミド標識をテストするために、pBR322の制限エンドヌクレアーゼ（REase）R.TaqI（5&#39;-TCGA-3 &#39;）を用?...

Watch this Scientific Journal Video about Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues at JoVE.com

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

DNA及びタンパク質は、配列特異的親和性またはDNAまたはタンパク質トランスフェラーゼおよび合成補因子類似体を用いた蛍光レポーター基で標識される。酵素の補因子特異​​性に応じて、アジリジンまたは二活性化補助因子アナログは、一次元または二段階標識のために用いられる。

メチルトランスフェラーゼと補因子類似体核酸およびタンパク質の配列特異的標識化

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

sequence-specific-labeling-nucleic-acids-proteins-with

Research

JoVE Journal

Biology

13.9K ビュー.  RWTH Aachen University.  DNA及びタンパク質は、配列特異的親和性またはDNAまたはタンパク質トランスフェラーゼおよび合成補因子類似体を用いた蛍光レポーター基で標識される。酵素の補因子特異​​性に応じて、アジリジンまたは二活性化補助因子アナログは、一次元または二段階標識のために用いられる。 

ビデオ: Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

一次組織の二つのチミジン類似体の免疫蛍光検出（CldUとIDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

生物学

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル- L</subキャプチャの複合質量分析法（CCMS）別> -ホモシステイン結合タンパク質

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

核酸の光度計微量定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

レーザーUncagableフルオレセインデキストランでゼブラフィッシュ細胞の系統ラベリング

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

マイクロボリュームバイオスペックナノ分光光度計を用いて核酸およびタンパク質の濃度測定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH /フラッシュラベルと細胞におけるテトラセンサータンパク質の画像解析

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

スルー代謝経路の確認と発見 13</supタンパク質構成アミノ酸の> C標識

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

3DNAを有する核酸構造の分析と構築

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...