この研究は、NIDCRの学内研究プログラムによってサポートされていました。

1。回路図の概要： このビデオでは、LIPSのアッセイを行うために必要な手順を示しています。抗原は、組換えウミシイタケルシフェラーゼ（RUC）-抗原の融合、および粗抽出物などのCOS1細胞で発現され得られ、精製せずに使用。 LIPSのアッセイは、マイクロタイターウェルの患者の血清を使用するCRUD 電子 RUC -抗原抽出物をインキュベートすることによって開始されます。抗体 - 抗原の混合物を、次いでIgG分子を捕捉するプロテインA / Gビーズを含む96ウェルフィルタープレートに移している。プロテインA / Gビーズを含むフィルタープレートを洗浄した後、RUC -抗原結合した抗体は、ルミノメーターで測定されるセレンテラジンの基板と光ユニットを添加することによって測定されます。 PART 1： シイタケ -抗原融合タンパク質の生産のためのプラスミドのトランスフェクション セットアップ：COS1細胞を標準組織培養のプロトコルを使用して、DMEM - 10％FCSで培養されています。 ウミシイタケルシフェラーゼ融合のためのプラスミドは、以前は1記載されている。これらのプラスミドのDNAはキアゲンからミディプレップキットを用いて調製される。収率は約1-3 mgのはずです。 -20℃で1000μg/ mlのストック溶液としてDNA濃度とストアを測定手順： <ol><li>トランスフェクション前日、分割されたCOS - 1 37新100への細胞プレートとインキュベート当たり約2 × 10 6時× 20 mmディッシュを℃に</li><li>次の日に、COS - 1細胞​​は80から95パーセントコンフルエントにする必要があります。各プラスミドDNA用ラベル1.5mlのポリプロピレンマイクロチューブは、トランスフェクトされる。 4℃で保存されているトランスフェ- 6トランスフェクション試薬は、室温にウォームアップ。 </li><li>各マイクロチューブにのOpti - MEMのメディアの94μlを添加する。次側の壁に触れることなくのOpti - MEM培地にFuGENE 6トランスの6μlを加える。 </li><li>室温で5分間混合物をインキュベートする。 </li><li>構築ウミシイタケルシフェラーゼ抗原の融合のためのプラスミドの1〜2μgのを（1mg/ml DNAのストックから）を追加します。混和し、次いで室温で15分間混合物をインキュベートする。 </li><li> COS1細胞の培地に均等にそれを滴下して細胞にDNA - FuGENE 6トランスフェ - のOpti - MEMのソリューションを転送する。 </li></ol> PART 2：収穫シイタケ -抗原の融合<ol><li>トランスフェクションの2日後、COS - 1細胞​​を回収している。これは、メディアを削除してからPBSを6mlで細胞を洗浄することで開始されます。 PBSをデカントした後、組織培養皿から離れて任意の残留PBSをピペット。 </li><li> 50mMトリス、pH7.5の、100mMのNaCl、5 mMのMgCl 2、1％トリトンX - 100、50％グリセロールおよびプロテアーゼ阻害剤（50 mlあたりの完全miniprotease阻害剤のカクテルの2錠で構成される冷溶解緩衝液1.4 mlを加え溶解バッファー）。セルの解体作業員による収穫の細胞は、すぐに氷上に2つの1.5mlマイクロチューブの各々にライセートの半分を譲渡する。 </li><li>ブランソン超音波処理150は、細胞が開いて分割するために使用されます。それぞれ、2,2および4の超音波処理の設定で5秒、5秒と5秒のための氷とパルスで細胞溶解液を含むマイクロ遠心チューブを置きます。 </li><li>最初のスピンの後、軽くチューブの側壁から緩やかに添付された残骸を削除するためにチューブを逆さに4℃で2つの4分間のスピン℃、12,500 rpmで細胞ライセートを遠心。第二スピンした後、氷上に新しいマイクロ遠心チューブに2本の管から、ペレットを中断することなく、上清を慎重に移す。 </li><li>ライセートの添加あたりの光ユニット（LU）を計算する。 LUを測定するために、新しいマイクロチューブにPBSを8μlのライセートの1μlを希釈する。直接希釈した混合物に1Xセレンテラジン基質100μlを加えるとすぐに5秒の読み取りとチューブルミノメーターを（20/20 Nターナーサイエンティフィック）を使用してチューブの発光を測定する。 </li><li> -80アリコートで時間の長い期間の1〜2日またはストアするため-20℃でRUC -抗原のライセート℃にて保存してください。 </li></ol> PART 3：セラマスタープレートの準備<ol><li>最初に（50 mMトリス、pH 7.5、100mMのNaCl、5 mMのMgCl 2、1％トリトンX - 100）96ディープウェルポリプロピレンマイクロタイタープレートの各ウェルにバッファー450μlを添加することにより血清のマスタープレートを作る。このステップでは、染料、フェノールレッドは、また将来の血清サンプルの追加とLIPSのアッセイの他のステップを監視するためのトレーサーとして動作する（最終濃度は、バッファーに0.2μg/ mlの場合）バッファに含めることができます。 </li><li>次の緩衝液A 450μlを含む別のウェルに各サンプルから血清50μlを追加するには、これは一般的に血清をマスタープレートの最後の二つのウェルに添加されていないバッファAの血清の1:10希釈ことに注意してくださいので、これは、バッファのブランクのために予約されています。 </li><li>マスタープレートを使用する前に、それは広範囲にローテータプラットフォーム上で（1-2時間）振とうする。マスタープレートの血清はSTAです。少なくとも1ヶ月BLE（または長い）4℃、蒸発を防ぐために正しく保存されている場合。 </li><li>後述のように、このマスタープレートを繰り返し、複数の抗原に対する血清中のプロファイリングのために削除される希釈血清10μlを提供します。大きくしたり小さくマスタープレートを使用することもできる。それが使用されていないパラフィルムでプレートをシール。 </li></ol>パート4：LIPSアッセイ<ol><li>ポリプロピレン96浅いウェルマイクロタイタープレートは、血清をテストするために使用されています。最初のステップでは、8チャンネルマイクロピペットを用いて96ウェルプレートの各ウェルにバッファー40μlを加える。 </li><li>次のマスタープレートから希釈血清（1μlの血清と同等）の10μlを取り、8チャンネルマイクロピペットを用いてワーキングプレートの各ウェルに直接追加してください。 </li><li> RUC -抗原の抽出物を含有するマスターミックスは、次の1 × 10 7ライトユニット（LU）を各ウェルに緩衝液50μlに追加されるように製剤化される。下の入力（わずか1 X 10 6）にも使用できますが、低ダイナミックレンジの結果をすることができます。各ウェルにこのマスターの混合物とRUC -抗原の混合物のピペットを50μlを加えます。 </li><li>室温で1時間ロータリーシェーカー上でインキュベートする。 </li><li>インキュベーション中に、新しい96ウェルフィルターHTSプレート（ミリポア、ベッドフォード、MA）の各ウェルの底にULTRALINKタンパク質PBSで/ Gビーズ（ピアースバイオテクノロジー、ロックフォード、IL）の30％懸濁液の5μlを追加。 </li><li> 1時間のインキュベーションの後、蛋白質8チャンネルマイクロピペットを使用して/ Gビーズを含む96ウェルフィルターHTSプレートに100μlのRUC -抗原抗体反応混合物を転送する。 </li><li>室温でさらに1時間のためのロータリーシェーカー上で96ウェルフィルタープレートをインキュベートします。 </li><li>次の真空マニホールドのフィルタープレートを洗浄する。各ウェルをPBS100μlで2回に続いて、バッファー100μlで8回洗浄する。これは、手動またはロボットピペッティングのワークステーションで実行できます。 </li><li>最後の洗浄に続いて、真空がオフになっています。フィルタープレートを取り外して、紙タオルやプレートの上部と下部に水分を除去することを確認してろ紙のスタックを使用して乾燥さ汚点。 </li></ol>第5部：測定発光とデータ分析セットアップ： ベルトルトのLB 960セントロマイクロプレートルミノメーターは、各ウェル単一のインジェクタを使用して発光を測定するために使用されます。マシンの電源がオンになると、インジェクタの洗浄サイクルを使用して蒸留H 2 Oでインジェクタをすすいでください。セレンテラジンを基質は、製造業者によって記載されたようプロメガシイタケの基板キットを用いて調製される。 1Xセレンテラジン基板ミックスの一般的に6mlの（すなわち、60μlのセレンテラジンの株式に加え、1Xバッファー6 ml）をプライミングマシンのために準備し、つの完全な96ウェルプレートを実行しています。プレートを分析する前に、ベルトルトのLB 960セントロマイクロプレートルミノメーターは、1Xセレンテラジンの基板で準備されている。基質を注入し、プレートを読み取るための設定を含むプログラムファイルを開きます。これらの測定では、セレンテラジンを基質50μlを注入した、プレートは発光の読み取り5秒に続いて、2秒間振とうする。 <ol><li>プレートルミノメーターはプレート全体を読み取る能力がありますが、板の部分的な読み取りは、（読み込みメニューの下）を選択することができます。プレートの読み取りを開始するプログラムを起動します。 </li><li>実行後、ルミノメーターでこぼれを防ぐために速やかにマイクロタイターフィルタープレートを取り外します。分析のためのExcel形式にMikroWinプログラムで生成されたデータをエクスポートします。 </li><li>我々は、2つの独立した実行から血清を評価することをお勧めします。その後、データは各サンプルのLUの力価の値を生成するために平均化されます。これらの値は、さらにバッファのブランクを差し引き、調整することができます。 </li><li>このようなカットを決定するなど、追加のデータ分析は、平均値と制御値の3または5の標準偏差を取ることによって計算することができます。 </li></ol>

<ol>
	<li>Burbelo, P. D., Goldman, R., Mattson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunoprecipitation+method+for+quantitatively+measuring+antibody+responses+in+clinical+sera+samples+by+using+mammalian-produced+Renilla+luciferase-antigen+fusion+proteins.">A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.</a> BMC Biotechnol. 5, 22-22 (2005).</li><li>Burbelo, P. D., Leahy, H. P., Iadarola, M. J., Nutman, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Four-Antigen+Mixture+for+Rapid+Assessment+of+Onchocerca+volvulus+Infection.">A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection.</a> PLoS Negl Trop Dis. 3, e438-e438 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+luminescent+profiling+of+anti-La+and+other+autoantibodies+in+Sj+gren&#x2019;s+syndrome.">Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sj gren&#x2019;s syndrome.</a> Autoimmunity. 139, 241-245 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antibody+quantification+and+generation+of+whole+proteome+antibody+response+profiles+using+LIPS+(luciferase+immunoprecipitation+systems).">Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems).</a> Biochem Biophys Res Commun. 352, 889-895 (2007).</li><li>Burbelo, P. D., Groot, S., Dalakas, M. C., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+definition+profiling+of+autoantibodies+to+glutamic+acid+decarboxylases+GAD65/GAD67+in+stiff-person+syndrome.">High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome.</a> Biochem Biophys Res Commun. 366, 1-7 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+luminescence+assay+for+autoantibodies+to+mammalian+cell-prepared+insulinoma-associated+protein+2.">A new luminescence assay for autoantibodies to mammalian cell-prepared insulinoma-associated protein 2.</a> Diabetes Care. 31, 1824-1826 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+human+herpes+simplex+virus+type+1+and+2+infections+by+luciferase+immunoprecipitation+system+assay.">Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.</a> Clin Vaccine Immunol. 16, 366-371 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+quantitative+serological+detection+of+anti-cytomegalovirus+(CMV)+antibodies.">Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.</a> Virol J. 6, 45-45 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-antigen+mixture+containing+v-cyclin+for+serological+screening+of+human+herpesvirus+8+infection.">Four-antigen mixture containing v-cyclin for serological screening of human herpesvirus 8 infection.</a> Clin Vaccine Immunol. 16, 621-627 (2009).</li><li>Ramanathan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luciferase+immunoprecipitation+systems+assay+enhances+the+sensitivity+and+specificity+of+diagnosis+of+Strongyloides+stercoralis+infection.">A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection.</a> J Infect Dis. 198, 444-451 (2008).</li></ol>

LIPSは、最小限のアッセイの最適化と、そのシンプルさのために、高品質のデータは通常、任意の抗原に2週間の下でLIPSを使用して生成することができますが必要です。最も時間のかかる手順がクローニングされ、RUC -抗原の融合を含む適切なプラスミド発現ベクターを生成する。これらのプラスミドが生成されると、単一または複数の抗原は、急速に上記のような唇でテストすることができます。それは、LIPSはそのようなその血清はまた、チューブの形式であるいは分注ロボットワークステーションの助けを借りて、マイクロタイターフィルタープレート2または吸引ろ過付きプレートウォッシャーでテストすることができる非常に汎用性であることに留意すべきである。この高度にスケーラブルなシステムとは、人間の自己抗原3またはウイルスの4の全プロテオームのパネルのプロファイリングを並列LIPSが可能になります。それは、LIPSによって生成される多くの異なった抗体のプロファイルは、治療10のコースに続く、ワクチンの監視診断3-9、抗原の発見9のに有用であると特定の疾患状態のため、一般的に公衆衛生のための広範な意味を持っている可能性が高いです。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>HiSpeed Plasmid Midi KIt</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1814443 </td>
<td>&nbsp;</td>
<tr>
<td>Complete, Mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836153001</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 96 DeepWell 1 ml plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120 </td>
<td>Deep well plate with lid and seal with Parafilm</td>
</tr>
<tr>
<td>Polypropylene microtiter plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120</td>
<td>&nbsp;</td>
<tr>
<td>HTS Filter plate</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSBVN1B50</td>
<td>&nbsp;</td>
<tr>
<td>Ultralink Immobilized Protein A/G</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>53133 (10ml)</td>
<td>&nbsp;</td>
<tr>
<td>Renilla Luciferase Assay System</td>
<td>&nbsp;</td>
<td>Promega </td>
<td>E2820</td>
<td>For 2000 assays</td>
</tr>
<tr>
<td>Centro microplate luminometer</td>
<td>&nbsp;</td>
<td>Berthold</td>
<td>LB 960</td>
<td>&nbsp;</td>		</tbody>
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		</div>

antibody profiling by luciferase immunoprecipitation systems (lips)

総合的に自己抗原と感染性病原体に対する抗体のプロファイルを理解し、定量化するための技術は、病気のメカニズムに新たな洞察をもたらすかもしれませんし、別の臨床的特徴と病因についての理解を深めるとともに病気をsubstratifyに新しいマーカーを明らかにすることがあります。我々は、感染症に関連する自己抗原と病原体抗原に対する患者血清の抗体応答をプロファイリングするためのルシフェラーゼ免疫システムを（LIPS）と呼ばれる高度に定量的な方法を開発した。 ELISA法とは異なり、高感度なLIPSは、簡単に普遍的な形式の異なる抗原に対する体液性血清学的応答プロファイルを調査するために実装し、動的な抗体価が6ログまで変化して生成され<sub&gt; 10</sub&gt;いくつかの抗原に対して。これらの研究では、抗原のパネルあるいはいくつかの病原体（すなわちHIV）の全体プロテオームに対する患者の体液性応答のLIPSによる定量的プロファイリングは、通常、ELISAによって単一の抗原をテストするより有益です。さらに、LIPSはまた高度に精製した抗原だけでなく、標準的なELISAのために必要な労働集約型のアッセイの最適化のステップを生成するために必要な時間と労力を排除。ここでは、容易に単一または複数の抗原に対する抗体応答をプロファイリングするための実行LIPSアッセイの技術的な側面を説明する詳細なプロトコルを提供しています。

Watch this Scientific Journal Video about Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS at JoVE.com

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

実行LIPS（ルシフェラーゼ免疫システム）の技術的な側面が説明されています。全体的なアプローチは、哺乳動物細胞でウミシイタケルシフェラーゼ（RUC）に融合した抗原をコードするキメラ遺伝子を発現する伴います。原油RUC -抗原の抽出液を調製し、精製することなく、抗体を定量化するために免疫沈降アッセイにおいて使用される。

ルシフェラーゼ免疫系による抗体のプロファイリング（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

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Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

26.7K ビュー.  National Institute of Dental and Craniofacial Research, National Institutes of Health. 実行LIPS（ルシフェラーゼ免疫システム）の技術的な側面が説明されています。全体的なアプローチは、哺乳動物細胞でウミシイタケルシフェラーゼ（RUC）に融合した抗原をコードするキメラ遺伝子を発現する伴います。原油RUC -抗原の抽出液を調製し、精製することなく、抗体を定量化するために免疫沈降アッセイにおいて使用される。

ビデオ: Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

マイクロ電極の構造によって生成されたAC動電現象

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

生物学

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

メチル化DNA免疫沈降

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM：フローサイトメトリーにより検出免疫沈降

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

複雑なサンプル中の特定のタンパク質のグリコシル化の多重化ハイスループットプロファイリングのための化学的にブロックされた抗体マイクロアレイ

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...