我々は、SAMの形成に有用な議論をメリッサGrunlanを認めます。この研究は、ロバートA.ウェルチ財団（A - 1585）で部分的にサポートされていました。

試薬の調製 PIPESバッファー（KOHでpH6.8に調整した80mMのPIPES、1mMのMgCl 2を 、1mMのEGTAを、）準備する。 -70分注し、店舗のグルコースオキシダーゼ、カタラーゼとグルコース-20 ° Cとタキソール℃の既存のプロトコルに応じて。-70 1,2分注しストアのチューブリン° C供給者が含まれている手順に従って。 リソグラフィーを用いたパターンのAu微小電極<ol><li>スクライブまたはウェハのカッターを使用して1 Cm&#39;と1.5cmの基板にシリコンウエハーをカット。それらをカバーし、5分間超音波処理するのに十分なアセトンをビーカーに配置することにより、基板をクリーニング。基板を乾燥させることなく、新鮮なアセトンでそれらを洗い流した後、直ちにイソプロパノールと乾燥を使用して窒素ですすいでください。 </li><li>希望の機能の大きさに応じて、標準、ポジ型のフォトリソグラフィーや電子線リソグラフィーを用いてクリーンな基板上にAu電極を作製。3パターンの形状を設計するとき、によってカバレッジを確保するために、100ミクロン以下の各電極パターンの直径を保つPEG SAM（次のセクションで説明）。電極（図1）でパッドを接続して1mmの線のパターンから500 mmまでの300を位置付け接触パッドを（〜300 mm 2）を含めます。フローチャンバーを組み立てるためのパターンの周囲にスペースを可能にするために基板の中央付近にリソグラフィパターンを置きます。 <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="図1" /> 図1サンプルの形状。してください<a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 </li><li>レジスト層のパターンの開発の後、ピンセット（図1）を使用して、基板のエッジに接触パッドからレジストのラインに傷を付けない。金属蒸着時には、スクラッチは、電極と基板の端との間の電気的接触が可能になります。好ましくは二つの情報源と成膜チャンバは、、金属層を堆積させるために使用されます。電極は、真空を破ることなく、Auの6nmのに続いてCrのは2nm（接着用）の堆積によって形成される。チャンバー内部に搭載した水晶振動子マイクロバランス法は、蒸着時のフィルムの厚さを測定するために使用することができます。 </li></ol> SAMを準備<ol><li>予熱および75換気の良い場所で℃のオーブン250mLのガラスビーカーにトルエン20mLを添加することによりシリル化ソリューションを準備する。その後、プラスチックピペットを用いてトルエンに1 mLのPEG -シラン（すなわち5％v / v）を追加。ピペッティング5〜10倍にしてから30秒間ピペットで撹拌することにより、PEG -シランよくで混ぜる。 </li><li>彼らは完全にPEG -シラントルエン溶液中に浸漬され、均等にビーカーの底部全体に等間隔ていることを確認しながら、上を向いてパターニングされた側とビーカー内でパターン化されたサンプルを置きます。オーブンでビーカーを置き、75で18〜21時間を焼く℃にSAMが形成されている間、対向の準備（下記参照）と、残りの手順に必要な追加のコンポーネントを組み立てます。 </li><li>オーブンで18〜21時間後、トルエンは粘性PEG -シラン残渣にパターン化されたサンプルを残して蒸発してください。ビーカーにトルエン20mlを追加して1-2分のサンプルを浸す。ビーカーからサンプルを削除してから、トルエン、イソプロパノールでリンスし、窒素を用いて最終的に乾燥した。この時点で、サンプルがきれいになっているはずですし、SAMの層は、肉眼では見えないはずです。 SAMのは、涼しい乾燥した条件（25℃、適度な湿度）下で保存した場合、最大2日まで使用可能なこの方法を準備した。表面の曇りや凹凸のある色のついた残のコーティングで失敗したSAMの形成の結果。 </li></ol>対向を作り上げる微小電極の堆積のために使用される同一成膜室のセットアップでは、22 × 50mmの1番か0番のガラスカバースリップ上のAuの4nmのに続いてCrの1nmを、堆積することによって対向電極を作る。対向はライトグレーやピンクの色合いとほぼ透明にする必要があります。それをきれいに保つためにシャーレにコーティングされた側を対向して保管してください。 MTSにチューブリンの重合<ol><li>未標識チューブリンとローダミンのチューブリンを解凍し、すぐに明るいMTを取得するために非標識する標識された1:2の比率で組み合わせる。数回上下にピペッティングして混ぜる。 37加熱した水浴中で20-40分のためのチューブリンを重合℃にMTの長さが長く、重合時間のために大きくなります。チューブリンが重合している間、PIPES緩衝液を含むマイクロ遠心チューブに20 mMにタキソールを追加することによって、PIPES -タキソールバッファを準備。上下に数回ピペッティングし、ボルテックスでよくタキソールを混合し、37℃で加熱した浴中にチューブを置きます℃に重合後、暖かいPIPES -タキソールバッファとシールドは1 / 100でMTを薄める軽いから。このようにして調製したMTSは、時間とさらに希釈することによって減らすことができるバンドルを、上に細長いなります。 </li><li> 30 mLの冷PIPES緩衝（0-4℃）、2 -メルカプトエタノール50％、カタラーゼ5mLのグルコースオキシダーゼ、5mLの、そして5mLのグルコースの5 mLを組み合わせることにより10倍の酸素掃気混合物（OS）を用意する。グルコース最後に追加する必要があります。各コンポーネントを追加した後に3-5回ピペッティングにより混和する。氷の上でOSの混合物を置き、それは約2時間のために効果的なままになります。 </li><li>準備MTSは1XのOSが混在してMTの重合が成功したことと、MTSはイメージングすることにより、蛍光励起の数分で安定していることを確認します。イメージングのためにこのようにして調製したMTSは、ローダミンの蛍光に適した励起/発光フィルターの組み合わせを使用してください。さらに1 × OSのミックスでPIPES緩衝とイメージングを使用して1 / 10 1 / 100にして、MTSを希釈することにより、イメージング、個々のフィラメントに最適な希釈率を決定する。 </li><li>両面テープの2つが離れて2〜3ミリメートルを置くと大（22 × 50 mm）と小型（22 × 22ミリ）のガラスカバースリップを挟むことにより、フローチャンバーを準備します。テープは簡単にガラススライド上に置き、カミソリの刃で切断して短冊状にカットされます。マイクロピペットを用いてフローセルにMTのいくつかのマイクロリットルを導入する。高倍率のイメージングのために、長い作動距離の目標は、イメージングフローチャンバーの上面のために必要となる場合があります。フローチャンバーの上面にカバースリップ/試料表面からの距離は、テープの厚さによって決定されると60〜100ミリメートルでなければなりません。 </li></ol>電気泳動を用いてパターンのMT <ol><li>電極は、テープ（図2）に垂直に接続するパスとストリップの間にあるような模様の、SAM被覆したサンプル全体に両面テープの二つの薄いストリップを配置することにより、電気泳動槽を準備します。このようなテープに触れる金属側に両面テープで対向電極を配置することを約3mm（図2）によるサンプルのオーバーハング対向のエッジ。被覆銅マグネットワイヤーのいくつかの15cmの長さをカットし、両端から絶縁体の1cmをはがします。 （これはマルチメータで確認できます）対向電極との電気的接触を避けるために注意しながら銀塗装の小さなドロップを使用して、基板の露出のAuのエリアにワイヤを取り付けます。銀塗料を使用して対向する2番目のワイヤを取り付けます。銀の塗料で接続されたワイヤは、さらにテープで試料に固定することができます。 <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="図2" /> 倒立型蛍光顕微鏡は図2。フローセルアセンブリ。してください<a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg">ここをクリックして</a>図2の拡大バージョンを参照すること。 </li><li> 1 ×最終濃度でOSと組み合わせてPIPES緩衝液を用いて所望の希釈でMTを導入する。 20倍の倍率で画像や電圧を印加する前に、シリコンウエハ上にパターンを見つけます。に応じて、フローチャンバーは、フローチャンバーの平面内の流体の流れとMTの移行を防止するために磨く爪を使ってシールすることができる。 </li><li>最大1 Vの電圧を印加し、MTの移行を観察。 MTの可逆的トラップが望まれる場合、低い電圧（0.5 V程度まで）を用いてもよいですが、これは横方向のドリフトを防ぐために、フローチャンバーを密閉する必要があります。マニキュアは、フローチャンバーの開口端をシールに適しています。約0.7 V以上の電圧では、いくつかMTの密着性は、それは弱いだろうけど、パターンに見られるかもしれない。約0.9 V以上の電圧で、MTの移行は速くなり、密着性が強くなります。約1.2 Vの上に誘導電気分解（ガスの気泡形成）のため、また、微小電極を破壊する可能性が大幅に増加します。移行は、顕微鏡の焦点の垂直面を調整することによって追跡することができます。正しく実施した場合、MTSは電極表面でローカライズされます。 </li></ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

MTの移行を容易にするため、その明るさと低バックグラウンドの蛍光を蛍光顕微鏡を用いて可視化されています。それが唯一の生体分子は緩衝液の適切な調整により、正味の電荷を運ぶために誘導されている必要がありますように、メソッドは、一般的に生体分子パターニングに適用可能である。このようなキャピラリー電気泳動に使用されるシリカの壁などの無帯電した表面が存在するた?...

第1ホウケイ酸ガラスカバースリップ（22 X 50 mm）をVWRから購入した。洗練された電子グレードのp -型&lt;111&gt;のシリコンウェーハを高純度銀の塗料が構造プローブ株式会社（カタログ＃5001 AB）から得られたアディソンエンジニアリング、（株）から購入した。絶縁ファインゲージの銅製マグネットワイヤーは、（〜0.08ミリメートル（0.003）直径）電子供給会社から入手することができます。

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

我々は、金の微小電極とシリコン基板上にポリ（エチレングリコール）（PEG）トリメトキシシラン自己組織化単分子膜（SAM）を形成するための手順を示します。 PEG - SAMは、単一のアセンブリ工程で形成し、シリコンと金の表面に生物付着を防止しています。 SAMは、標準、ポジ型のリソグラフィでパターン化されたコートの微小電極に使用されます。例として、微小管を使用して、我々は、可逆的な方法でSAM被覆電極に電気泳動を誘導するDC電圧を印加してください。フローチャンバーは、イメージングのために電気泳動と微小管のパターニングを使用されています<em&gt;その場で</em&gt;落射蛍光顕微鏡を用いて。それは標的分子を固定化する電気泳動を採用し、したがって、特定の分子の相互作用を必要としないとして、このメソッドは、一般的に生体分子パターニングに適用可能である。さらに、それは直接リソグラフィ技術を使用してパターンにSAM分子を試みるときに発生する問題を回避することができます。電子ビームリソグラフィーとの互換性は、このメソッドは、ナノスケールのパターンの生体分子に使用できるようになります。

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

我々は、金の微小電極とシリコン基板上にポリ（エチレングリコール）自己組織化単分子膜を（PEG - SAM）を形成するための手順を示します。 PEG - SAMは、単一工程で形成し、シリコンと金の表面に生物付着を防止しています。電気泳動は、ナノスケールにパターニング生体分子のためにダウンして使用されます。

パターニングの生体分子の微小電極上に自己組織膜を汚

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

antifouling-self-assembled-monolayers-on-microelectrodes-for

Research

JoVE Journal

Biology

11.4K ビュー.  Texas A&M University (TAMU). 我々は、金の微小電極とシリコン基板上にポリ（エチレングリコール）自己組織化単分子膜を（PEG - SAM）を形成するための手順を示します。 PEG - SAMは、単一工程で形成し、シリコンと金の表面に生物付着を防止しています。電気泳動は、ナノスケールにパターニング生体分子のためにダウンして使用されます。

ビデオ: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

光学的に透明な酸化インジウムスズ電極上にパターニングセル

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

生物学

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

バイオフリップチップを用いた胚性幹細胞のパターニング

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定化生体分子の単一分子蛍光測定のための自動化システム

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成体マウスの心筋細胞の単一細胞転写プロファイリング

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

プロテインWISDOM：ベンチ用<em&gt;インシリコで</em&gt;<em&gt;デノボ</em生体分子の&gt;デザイン

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

エレクトロポ生きた微生物で内在化および蛍光生体分子の観測

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

外酸生産の測定と解析は、解糖速度を決定するために

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

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 ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

部位特異的タンパク質のリジンのアセチル化のサクシニル化化学量論組成データに依存しない取得質量分析法による定量

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

セノ治療薬同定のためのSA-β-ガラクトーシダーゼベーススクリーニングアッセイ

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...