NU - ELISA法の開発は、NIHの助成金RO1AG023687によってサポートされていました。

1。哺乳動物細胞培養 NU - ELISAは、培養で増殖することができます任意の哺乳動物細胞の種類に実行することができます。そこで我々は、いくつかの異なる抗体（ABS）でアッセイを可能にする、ヌクレオソームがいくつかの同じロードされたELISAプレートを準備するのに十分な量で単離することができるように、細胞の大規模なバッチに適度な準備をすることを好む。以下の培養スケールでは十分な材料を提供します。 マウスembyronic幹細胞は、フィーダー細胞なしで細胞の1つまたは2つの15cmのプレートを成長させる。線維芽細胞の場合は、合流するために5〜10 15 cmのプレートを成長させる。 2。原子核のアイソレーション注：すべてのステップが示されている場合を除き、あらかじめ冷却した緩衝液で氷の上です。 <ol><li>プレート当たり3 mlのトリプシンで細胞をトリプシン処理、結合、および氷冷PBS /酪酸の20 mlを加える。 5分間1000rpmで遠心する。 </li><li> 10ミリリットルのPBS /酪酸、5分間1000rpmで遠心分離で細胞を再懸濁します。 </li><li>プロテアーゼ阻害剤（Sigma - Aldrich社P - 8340）4 mlの溶解バッファーで再懸濁します。氷の上でタイプBの乳棒で20ストロークを均質化するダウンス。 </li><li>で10分間4℃で2000グラムで遠心分離（上清は、このプロトコルのために必要されていない細胞質が、含まれているが、必要であればそれは他の用途のために保存することができます）。 </li><li> 2ミリリットルの氷冷洗浄緩衝液C（プロテアーゼ阻害剤を含む）でペレットを再懸濁します。 </li><li>レイヤ5ミリリットル、30％ショ糖クッション上に再懸濁した物質は、その後、スイングバケットローターで5分間2400グラムで遠心。核はクッションを通って移動し、破片は、インターフェイスのままです。 </li><li>慎重にすべての液体のボリュームを削除し、250μlの氷冷洗浄緩衝液C +プロテアーゼ阻害剤の核を再懸濁します。 </li></ol> 3。 その場でミクロコッカスヌクレアーゼ（MNase）消化のでヌクレオソームの単離我々はクロマチンが上清に遊離無傷ヌクレオソームを駆動する低張処理することにより、MNaseでそれらを導入することで、核内にその場で消化されているプロシージャを使用してください。 <ol><li>サンプルに3μlの0.1 M CaCl 2を追加。 37℃のヒートブロックに入れ。 37℃の温度を仮定することができます。 </li><li> 2台のMNaseを（ミクロコッカスヌクレアーゼ、シグマアルドリッチ、MNaseバッファの2units/10μlで溶解）を追加、その後37℃のためにインキュベートしますピペットチップを使用して頻繁に混合しながら12分間のCを、。 </li><li>反応を停止するには、0.5MのNa - EDTA、pH8.0の6μlを添加する。氷上に置く。 </li><li> 4分間2000グラムで遠心分離、上清を捨てる。 300μlの0.2mMののNa - EDTAでペレットを再懸濁する。時折優しいペッティングで1時間氷上で保存する。 （これらの低張条件は核から遊離ヌクレオソームを解放）。 </li><li> 4℃で4分間3000gで遠心する℃、氷の上で、自由なヌクレオソームを含む上清を、保存します。 </li><li> 300μlの0.2mMののNa - EDTAで再び沈殿を再懸濁します。時折優しいペッティングで1時間氷上で保存する。 </li><li> 4℃で4分のために3000グラムで遠心分離℃を上清を保持し、手順5からの最初の上清と組み合わせる。結果mononucleosome製剤は-80℃で分注し、保存することができます。 </li></ol>注：クロマチンの量が粗雑に10μlのサンプルの260nmの吸光度を測定することによって定量することができる水990μlに追加。 260 = 10（1 / 100希釈を調整後）クロマチンの約1mg/mlに相当する。主に長さが146塩基のDNAが含まれていなければなりませんMNase消化の質と程度を、監視するために彼らのDNAの含量のためにも、上記の手順の間に、少量のサンプルが保持される場合があるので、後で分析する。ラダリング型は、不完全なMNase消化の指標である。 注：図1は、核の分離とMNase消化の手順の図示した要約が含まれています。 4。ヌクレオソーム- ELISA（NU - ELISA） 我々は、96ウェルELISAマイクロタイタープレート上に固定されているネイティブそのままnuclesosomesを含有する画分にPTMSを検出する。各サンプルについて、我々は2倍希釈系列を作り、これらは三連のウェル上にコーティングされています。 <ol><li> 4 °50μl/ウェルのコーティング緩衝液で希釈したヌクレオソームとC。一晩コートMaxiSorpプレートヌクレオソームの提案されたシリアル倍の希釈液を底部で唯一のコーティング緩衝液（0μgのクロマチン）を、0.1μgの、濃度0.05μg、0.025μgの、0.0125μgの、0.00625μgの、0.00313μgの、0.00156μgを追加することによって、下の行に一番上の行から調製される行。 （注：プレートのカバーは、このステップのために必要です。） </li><li>午前中に、プレート洗浄機で10分間の合計のために室温（RT）で200μl/ウェルPBS/0.5％ツイーン20でプレートを4回洗浄する。 </li><li> 100μl/ウェルPBS/0.05％のTween - 20 / 5％BSAで室温でブロック1時間。 </li><li>活発に反転プレートを振とうしてブロッキングバッファーを除去するシンク上。カバーと-20℃でプレートを保存したり、次の手順に進みます。 </li><li> PBS/0.05％ローテーターで室温で1時間インキュベートのTween - 20 / 5％BSA、50も希釈/μL1:1000の体積（または必要に応じて）に1 · ABSを追加。 </li><li>室温とプレートウォッシャーで10分の合計のための200μl/ウェルPBS/0.5％ツイーン20でプレートを4回洗浄する。 </li><li>ホースラディッシュペルオキシダーゼ（HRP）標識2 °腹筋、PBS/0.05％ローテーター上で時間室温でのTween - 20 / 5％BSAで（または必要に応じて）1:5000に希釈を追加。 </li><li> 200μl/ウェルPBS/0.5％ツイーン20でプレートを4回洗浄する。各洗浄には、プレートウォッシャーを使用して室温で10分の合計です。 </li><li>室温で10分間各ウェルにTMB基質50μlを加えてプレートを開発する。 H 2 SO 4を50μlを添加することにより/よく2Nの反応を停止し、プレートを450 nmのを読んで。 （ヒント：前の読書吸光度に井戸の気泡を放​​散するためにプレートのローターを使用して2分間1500rpmで遠心分離）。 </li><li>定量的および統計的分析のためのスプレッドシートファイルへの読み取り値をエクスポートします。 </li></ol>試薬<table border="1"><tbody><tr><td> PBS /酪酸 </td></tr><tr><td> 135 mM NaClを</td></tr><tr><td> 2.5mMのKClを</td></tr><tr><td> 8ミリのNa 2 HPO 4 </td></tr><tr><td> 1.5mMのKH 2 PO 4 </td></tr><tr><td>の10mMのNa -酪酸</td></tr></tbody></table><table border="1"><tbody><tr><td> 溶解バッファー </td></tr><tr><td> 250mMスクロース</td></tr><tr><td> 10mMトリス- HCl、pH7.4の</td></tr><tr><td>の10mMのNa -酪酸</td></tr><tr><td> 4mMのMgCl 2の </td></tr><tr><td> 0.1％トリトンX - 100 </td></tr></tbody></table><table border="1"><tbody><tr><td> 洗浄バッファーC </td></tr><tr><td> 250mMスクロース</td></tr><tr><td> 10mMトリス- HCl、pH7.4の</td></tr><tr><td>の10mMのNa -酪酸</td></tr><tr><td> 4mMのMgCl 2の </td></tr></tbody></table><table border="1"><tbody><tr><td> ショ糖クッション </td></tr><tr><td>洗浄バッファーCで30％（w / v）スクロース</td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocalヌクレアーゼ緩衝剤 </td></tr><tr><td> 5mMのナポ4緩衝液、pH 7.0の</td></tr><tr><td> 0.025ミリメートルのCaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> コーティング緩衝液 </td></tr><tr><td> 80ミリリットルの溶液A + 170ミリリットル溶液B + 250ミリリットルのdH 2 O </td></tr><tr><td>解決策：0.2 M Na 2 CO 3の </td></tr><tr><td> B液：0.2 M NaHCO 3を </td></tr></tbody></table> 5。代表的な結果 NU - ELISA法を用いて、複数の同一のプレートのシリーズはmononucleosomesの形で準備したクロマチンで読み込むことができる準備されています。我々は通常、それぞれが異なる抗PTM特異的抗体（ABS）でプローブすることができるように同一にロードされたプレートのシリーズを用意。我々はまた、常に、その修正の状態合計クロマチンのローディングコントロールに関係なく、ヒストンを検出する抗体でプローブすることができますプレートを準備。このコントロールは、後で定量的に異なる試料から調製したヌクレオソームのPTM内容を比較するために不可欠です。我々はこのアプローチを使用して生の吸光度値を修正特定のクロマチンサンプル内PTMSのレベルを定量するために：まず、ないクロマチンを（背景は通常無視できる）含まないコントロールウェルから得られた値を使用して、任意のバックグラウンド信号を引きます。私たちは、その後、その修正の状態に関係なく、ヌクレオソームを検出する抗体をアッセイした同じロードされたプレートから入手したNU - ELISAの結果にPTM -特定の信号を標準化する。 （我々はこの目的のためにヒストンH2AまたはH2Bに特異的なポリクローナルAbsを使用している）。私たちは、その後、クロマチンの各希釈に平均値と分散を決定し、そしてELISAアッセイの直線部分から得られたデータを使用してください。 NU - ELISAの数学と統計分析の詳細な例は、以前は次の4報告されている<img src="/files/ftp_upload/2593/2593fig1.jpg" alt="図1"/> 図1。 NU - ELISAの手順の概略diagam。 A.哺乳動物細胞を収穫している、B.の核はダウンスhで細胞から分離されていますomogenization、C.は、細胞を遠心分離後、30％w / vのスクロースクッション、D.の上にロードされている混乱、核はペレット内に堆積され、E、FのクロマチンはMNaseによって主にmononucleomesにその場で消化され、G、H 、私 。可溶性mononucleosomesは低張処理し、残留核物質の繰り返し遠心分離によって抽出されます。J.の Mononucleosomesは異なる試料から（ラベルはSAMP。この場合は1と2）は同じロードされたプレートのシリーズでマイクロタイターウェルにコーティングして尋問されています合計クロマチンの負荷を決定するためにPTM -固有の腹筋とPTM -独立の腹筋。そのPTM内容が異なる2つのサンプルに差動信号の検出レベルのK.の描写が、まだ、抗H2B免疫反応性によって判断、同じような全体的なクロマチンのコンテンツを持っている。

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

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NU - ELISAは、特定の細胞型内に存在するヌクレオソームPTMSのグローバルステータスを確認する方法を提供する。 NU - ELISAの研究は世界的なヌクレオソーム変更の状態が発散細胞型4の比較で異なることが示されている。細胞はトリコスタチンA -またはときにマウスembyronicの幹細胞は4を差別化しているなどのエピジェネティックな調節剤にさらされているときに加えて、NU - ELISA PTMのプロファイルが変更されます。方法はまた、ヒトES細胞5のクロマチンを研究するために正常に適用されています。 NU - ELISAは、現在の形で、細胞のエピゲノムの合計内に存在するPTMSの唯一のコンポジット署名を検出することができるに注意する必要があります。これは、特定の遺伝子座における特定のPTMの分布を決定することができるゲノムワイドなクロマチン免疫沈降、と著しく異なります。 NU - ELISAの手順の最初のステップは、哺乳動物の核6,7からmononucleosomesを分離する従来の方法を適合させたもの。これらの適応の方法は、哺乳動物細胞から高品質の無傷mononucleosomesを得るために好都合な方法を提供し、得られた画分は、クロマチンコンテンツで包括的ですが、追加の核物質が大量に含まれています。特定の腹筋が使用されているので、混入非ヌクレオソーム材料は、高い純度を必要とする量産仕様のアプローチとは異なり、NU - ELISAアッセイで忍容性は良好です。しかし、NU - ELISAは、西部が西部劇とヌクレオソームPTMSの検出のための質量分析に優れた代替手段を提供するので、4ブロッティングよりも高感度かつ定量的です。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>ミクロコッカスNucleae </td><td>シグマ</td><td> N3755 </td><td> -20℃で保存し、2U/10μlバッファのアリコートを作る</td></tr><tr><td>ヌンクMAXISORPマイクロタイタープレート</td><td>フィッシャー</td><td> 12-565-135 </td><td>接着プレートカバーもフィッシャーを通じて注文することができます</td></tr><tr><td>自動化されたプレートウォッシャー</td><td></td><td></td><td></td></tr><tr><td> 1ステップTMB - ELISA基質</td><td>ピアース</td><td> 34028 </td><td> 4℃で使用前に室温に事前暖かいまたは仕様書で指示</td></tr><tr><td>マイクロタイタープレートリーダー</td><td> Bio - Rad社製マイクロプレートリーダー（モデル：680） </td><td></td><td>ほとんどの比色プレートリーダーのモデルが適しています。 </td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

真核生物のゲノムは、DNAとタンパク質の両方を含むクロマチンとして存在する。クロマチンの基本単位は2つのヒストンH2A、H2B、H3、およびH4 1のそれぞれに関連付けられているDNAの146塩基対を含むヌクレオソーム、です。ヒストンのN末端尾部には、リジンとアルギニンに富み、アセチル化、メチル化、および他の翻訳後修飾（PTMS）によって転写後に変更されます。ヌクレオソームのPTMの構成は、このような性質2,3におけるエピジェネティックな遺伝子調節のモードを提供し、関連するDNAの転写活性に影響を与えることができます。我々はヌクレオソームELISA（NU - ELISA）は定量的に細胞から抽出したヌクレオソームのグローバルPTMの署名を決定するという方法を開発した。 NU - ELISAはウェスタンブロット法よりも感度が高く定量的であり、そして特定の細胞型のepiproteomic状態を調べるために便利です。このビデオジャーナルの記事では、NU - ELISA解析を実行する詳細な手順を示しています。

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

ヌクレオソームELISA（NU - ELISA）は、ネイティブ、無傷のヌクレオソームの調製品で、翻訳後修飾のグローバルなパターンを検出する高感度かつ定量的な方法です。これらの変更は、メチル化、アセチル化、および特定のヒストンのアミノ酸残基でリン酸化が含まれており、それ故にNU - ELISAは、特定の細胞型の総合的なクロマチンの修飾状態のグローバルプロテオミクスアッセイを提供する。

ELISAによるネイティブ無傷ヌクレオソーム上の翻訳後修飾の検出

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

detection-post-translational-modifications-on-native-intact

Research

JoVE Journal

Biology

17.2K ビュー.  Stanford University. ヌクレオソームELISA（NU - ELISA）は、ネイティブ、無傷のヌクレオソームの調製品で、翻訳後修飾のグローバルなパターンを検出する高感度かつ定量的な方法です。これらの変更は、メチル化、アセチル化、および特定のヒストンのアミノ酸残基でリン酸化が含まれており、それ故にNU - ELISAは、特定の細胞型の総合的なクロマチンの修飾状態のグローバル?...

ビデオ: Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

ザイモグラフィーによる機能マトリックスメタロプロテイナーゼの検出

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

生物学

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

偏上皮細胞へのプラスミドのマイクロインジェクションによる低分子量GTPaseの機能を解析する

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

植物の葉におけるヒストン修飾の検出

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物タンパク質複合体の翻訳後修飾の同定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

enChIPによって特定のゲノム領域と関連する分子の同定の単離

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

無傷の動脈の内皮層における一酸化窒素とスーパーオキシドのエン顔検出

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

トマト毛状根の発生によるDNAの修飾のハイスループットCRISPRベクター構築と特性

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

ヒストンの抽出からデータ解析まで：ボトムアップ質量分析法を用いてヒストンの翻訳後修飾の解析のための完全なワークフロー

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

無傷の植物をブランチングすることによって、または抽出物の熱処理によりタバコ宿主細胞タンパク質の除去方法の比較

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...