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1。細胞の調製<ol><li> 5％CO 2の加湿雰囲気で10％ウマ血清、37℃で培養を維持すると補足したダルベッコ改変イーグル培地（DMEM）で下垂体AtT20細胞を成長させる。 </li><li>標準的なトリプシン処理プロシージャを使用してすべての5〜7日の細胞を継代培養により培養を維持しています。 </li><li>コー​​ト、黒、透明底のウェル、ポリ-L-リジンの50μL、96ウェルプレート。井戸はインキュベーター内で30分間乾燥させます。 </li><li>ウェル当たり30,000細胞の密度で3-4日間培養器内のセルを格納し、200μLの培地を含有する96ウェルプレートのプレートの細胞を。 </li><li>徐々に細胞単層から培地を吸引するために、一方の端に真空ともう一方の端に200μLピペットの先端に接続された栓を4リットル瓶で構成され、誤嚥のセットアップを使用しています。 </li><li>通常の緩衝液（132 mM塩化ナトリウム、5 mMのKClを、1メートルの200μLで細胞を洗浄MのCaCl 2、1mMのMgCl 2、5mMのデキストロース、5mMのHEPES、NaOHでpH 7.4）。 </li><li>各ウェルに膜電位感受性色素（MPD）DiBAC 4（3）またはHLB 021から152（5μM）を含む緩衝液200μLを追加し、37℃で40分間細胞をインキュベート℃に</li></ol> 2。試験化合物および細胞治療の準備<ol><li> Versette（サーモフィッシャー）自動液体ハンドリングシステムを使用して、化合物ライブラリーからの試験化合物の希釈（DMSO中ストック= 10 mM）を（96ウェルフォーマットで）を準備します。 </li><li>インキュベーターから、At​​T20細胞を含む、96ウェルプレートを取り外し、プレートを室温に戻すことができます。古いバッファをオフに吸引し、細胞へのMPDを含む新鮮な緩衝液を適用します。 </li><li>液体ハンドリングシステムは、様々な試験化合物の濃度（10 nMの10μMまで）（2から20μL）と制御ソリューション（0.1％DMSO）を（緩衝液containiに適用される使用NG MPD）細胞を含む96ウェルプレートへ。 </li><li>蛍光測定の前に試験化合物を5分間、細胞をインキュベートします。 </li></ol> 3。 GIRKチャネルの活性化、蛍光測定と解析<ol><li>バイオテックSynergy2蛍光プレートリーダーに96ウェルプレートを挿入し、それぞれ、520および560nmの励起および発光波長で蛍光バックグラウンド信号を得る。 </li><li> Synergy2インジェクターシステムを使用して、GIRKチャネルをアクティブにする（総量= 220μL）をウェルにGPCRリガンド（ソマトスタチン= 200 nM以上カルバコール= 10μM）またはコントロール溶液（緩衝液のみ）（20μL）を適用します。それぞれ520および560 nmの励起および発光波長で300秒のサンプリング期間を10秒間隔でデータポイントを収集します。 </li><li>時間経過とバイオテックGEN5ソフトウェアを用いた蛍光変化のピーク振幅を取得し、さらに分析するためにデータをExcelにエクスポートします。 </lI&gt; <li>アッセイの信頼性を決定するためにZ &#39;因子を計算します。 Z &#39;因子の決定のために使用されるプレートは、2行正（GPC​​Rリガンド）と負（バッファーのみ）の各コントロールが含まれていました。 Z &#39;因子は次のように定義されています。Z&#39; = 1 - （3σP +3σN）/ |μのP - μN |μP＆μNは正の制御と負の制御信号の手段である、、、σ P＆N、σは正の制御とそれぞれ負の制御信号、6の標準偏差である。 0.5から1.0の範囲でZ &#39;因子は、アッセイの品質が6に優れていることを示します。 0.5以下Z&#39;-因子とプレートは分析から除外されます。 </li><li>蛍光シグナルを調節する化合物は、内向き整流K +チャネルの特異性を確認するのBa 2 +の存在またはtertiapin-Qで再検査されています。 </li></ol> 4。 Representative結果 GIRKチャネルアッセイを用いて測定した蛍光シグナルの例を図2に示されています。 AtT20細胞にGPCRリガンドのソマトスタチン（200 nm）の添加は、HLB 021から152蛍光シグナル（ 図2）が急速に、時間依存性の減少を引き起こした。対照的に、コントロール溶液の添加は、時間とともに（ 図2）ベースラインに戻ったことを蛍光の小さな瞬間的な上昇を作り出した。制御およびソマトスタチンソリューションを注入し、96ウェルプレートのピーク蛍光値の比較は0.5から0.7の範囲でZ &#39;因子を与えた。さらに、定量化のため、制御レコードはソマトスタチンレコードと（ 図2）分析結果のソマトスタチン敏感な信号から減算されました。 アッセイは迅速にGIRKチャネルを調節する薬剤を同定する方法を提供する。 GIRKチャを阻害する、例えば、薬物nnel細胞からK +流出を防止することにより、蛍光のGPCRリガンド媒介減少を減らす必要があります。などのブロックGIRKチャネル7その毒素、tertiapin-Qによる細胞の処理は、 図3に示すように、ソマトスタチンを介した蛍光変化の抑制を作り出した。プロパフェノン、心臓8のブロックGIRKチャネルを抗不整脈薬はまた、蛍光の変化（ 図4）を阻害した。アッセイはまた、GIRKチャネルの活性化を識別するための役に立つかもしれません。ソリューション（P&gt; 0.5）を制御すると比較した場合しかし、エタノールのアプリケーション（100＆200 mM）を、GIRKチャネル2,3の活性化は、蛍光シグナルの有意な変化を引き起こさなかった。 <img src="/files/ftp_upload/3850/3850fig1.jpg" alt="図1"/> 図1。GIRKチャネル蛍光アッセイの実験的デザイン。 AtT20細胞はmembranを含む緩衝液中でインキュベートされる電子電位感受性蛍光色素（D）。色素分子は、細胞に入ると細胞内のタンパク質（上のパネル）に結合すると蛍光灯になります。そのGPCRへ（SOM）ソマトスタチンの結合は、GIRKチャネル（下パネル）の活性化を引き起こすG阻害タンパク質（G i）を刺激します。 K +外細胞のその後の流出は、膜電位は細胞を終了するには、より負と色素分子になるようになります。結果として、蛍光シグナルの減少（下パネル）。 <img src="/files/ftp_upload/3850/3850fig2.jpg" alt="図2"/> ソマトスタチンまたはコントロール溶液の添加時にAtT20細胞内で経時的に測定図2。代表HLB 021から152蛍光シグナル。蛍光強度（F / F O）の比率は（F O）広告する前に測定されたベースライン信号によるソマトスタチンの有無（F）（またはコントロール溶液）で信号を割ることによって算出したソマトスタチンのdition（またはコントロール溶液）。各点は平均値±SE 5から6の井戸で得を表しています。ソマトスタチンまたはコントロール溶液は、時間ゼロ（↓）で追加されました。コントロール溶液の添加は、蛍光シグナルの小さな一時的な増加を引き起こした。これは、溶液の注入によって引き起こされる温度変化に起因する。 <img src="/files/ftp_upload/3850/3850fig3.jpg" alt="図3"/> 図3。tertiapin-Q（500 nm）でAtT20細胞の治療は、ソマトスタチン媒介に結合し、GIRKチャネルを遮断することによって蛍光シグナルの減少を抑制する。各点は平均値±4から6の井戸で得られたSEを表しています。ソマトスタチンは、時間ゼロ（↓）で追加されました。 <img src="/files/ftp_upload/3850/3850fig4.jpg" alt="図4"/> 図4プロパフェノン（20μM）とAtT20細胞の治療はまたのソマトスタチン媒介の減少を抑制する蛍光シグナル。各点は平均値±SE 5から6の井戸で得を表しています。ソマトスタチンは、時間ゼロ（↓）で追加されました。

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利害の衝突が宣言されません。

膜電位感受性蛍光色素がイオンチャネルを調節する薬物9,10を識別するために使用されているが、これはニューロンのGIRKチャネル創薬への応用の最初の報告である。ここに提示GIRKチャネルの蛍光アッセイは、リガンド依存性K +チャネルのスクリーニングのための、高速で信頼性の高いリアルタイムメソッドを提供します。アッセイは、内因性のチャネルを発現している外因性...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> DMEM </td><td> CellGro </td><td> 10から013 </td></tr><tr><td>ウマ血清</td><td>インビトロジェン</td><td> 16050-114 </td></tr><tr><td> 96ウェルプレート</td><td>コー​​ニング</td><td> 3603 </td></tr><tr><td>ポリエルリジン</td><td> Sigma-Aldrich社</td><td> P4707 </td></tr><tr><td>ソマトスタチン</td><td> Sigma-Aldrich社</td><td> S9129 </td></tr><tr><td>カルバコール</td><td> Sigma-Aldrich社</td><td> C4382 </td></tr><tr><td> HLB 021から152 </td><td> AnaSpec </td><td> 89300 </td></tr><tr><td> Versette自動化された液体ハンドラ</td><td>サーモフィッシャー</td><td> 650から01 </td></tr><tr><td> Synergy2蛍光プレートリーダー</td><td>バイオテック</td><td></td></tr><tr><td> GEN5分析のoftware </td><td>バイオテック</td><td></td></tr></tbody></table> 表1特定の試薬 ​​や機器の表。

a fluorescent screening assay for identifying modulators of girk channels

Gタンパク質依存性内向き整流K +（GIRK）チャンネルはホルモンや神経伝達物質の広い範囲の細胞のメディエーターとして機能し、脳、心臓、骨格筋、内分泌組織の1,2で表されます。 GIRKチャネルは、それらの細胞膜結合型のリガンド（神経伝達物質、ホルモン、薬物など）、Gタンパク質共役型受容体（GPCR）の結合後に有効になります。この結合は、その後に結合し、GIRKチャネルを活性化するGタンパク質（G iとG o）の刺激を引き起こす。一度GIRKチャネルは、Kの動きを可能にする+外より負になるための静止膜電位を引き起こす細胞からオープンしました。その結果、ニューロンのGIRKチャネルの活性化は、自発的活動電位の形成を減少させ、興奮性神経伝達物質の放出を阻害する。心の中で、GIRKチャネルの活性化は、それによって心拍数を遅くするペースメーカー活性を阻害する。 <pクラス= &quot;jove_content&quot;&gt; GIRKチャネルは、神経因性疼痛治療、薬物中毒、不整脈や他の疾患3の新しい治療薬の開発のための新たな目標を表すものです。しかし、これらのチャネルの薬理学は、主に未踏のままです。抗不整脈薬、抗精神病薬や抗うつ薬を含む薬物の数は、GIRKチャネルをブロックするが、この阻害は選択ではなく、相対的に高い薬物濃度を3で発生します。 ここでは、GIRKチャネルの新たなモジュレーターを同定するためのリアルタイムスクリーニングアッセイを説明します。このアッセイでは、GIRKチャネルを発現神経AtT20細胞は、例えば、ビス- （1,3 - dibutylbarbituric酸）トリメチンオキソノール[DiBAC 4（3）]またはHLB 021から152（ 図1のような膜電位感受性蛍光色素を使用してロードされ。）色素分子は、細胞（ 図1）に強い蛍光を、次の取り込みになります。治療GPCRリガンドと細胞のGIRKチャネルを開くために刺激する。細胞外の結果K +流出は、膜電位がより負と減少する蛍光シグナル（ 図1）になるようになります。したがって、GIRKチャネルを介してK +流出を調節する薬剤は蛍光プレートリーダーを用いてアッセイすることができます。他のイオンチャネルスクリーニングアッセイなど、原子吸光分析法4または放射性トレーサー解析5と異なり、GIRKチャネル蛍光アッセイは、高速リアルタイムかつ安価なスクリーニング法を提供します。

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Gタンパク質依存性内向き整流Kと相互作用する薬剤を同定するためのリアルタイムスクリーニングの手順<sup&gt; +</sup&gt;（GIRK）チャンネルが記載されています。アッセイは、GIRKチャネル活性を測定する膜電位感受性蛍光色素を利用しています。この手法は、セルの行数での使用に適応可能である。

GIRKチャネルのモジュレーターを同定するための蛍光スクリーニングアッセイ

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

a-fluorescent-screening-assay-for-identifying-modulators-girk

Research

JoVE Journal

Biology

14.3K ビュー.  University of South Carolina, School of Medicine. Gタンパク質依存性内向き整流Kと相互作用する薬剤を同定するためのリアルタイムスクリーニングの手順 +（GIRK）チャンネルが記載されています。アッセイは、GIRKチャネル活性を測定する膜電位感受性蛍光色素を利用しています。この手法は、セルの行数での使用に適応可能である。

ビデオ: A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

多重抗体スクリーニング法を用いたLuminex XMAPアッセイへの捕捉ELISAの変換

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向き整流カリウムチャネルの小分子モジュレーターのためのハイスループットスクリーニング

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

プロテオリポソー​​ムベースの流出アッセイのClの単一分子特性を決定する<sup&gt; -</sup&gt;チャネルとトランスポーター

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

多重化アプリケーションのための蛍光タンパク質との遺伝バーコーディング

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

蛍光クリアランスアッセイを用いてゼブラフィッシュ腎臓機能の評価

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...