この作品は、ルミネックス·コーポレーションによって資金を供給された。

<ol>
	<li>Fulton, J. R., McDade, R. L., Smith, P. L., Kienker, L. J., Kettman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+multiplexed+analysis+with+the+FlowMetrix+system.">Advanced multiplexed analysis with the FlowMetrix system.</a> Clinical Chemistry. 43, 1749-1756 (1997).</li><li>Carson, R. T., Vignali, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantation+of+15+cytokines+using+a+multiplexed+flow+cytometric+assay.">Simultaneous quantation of 15 cytokines using a multiplexed flow cytometric assay.</a> J. Immunol. Methods. 227, 41-52 (1999).</li><li>Peck, D., Crawford, E. D., Ross, K. N., Stegmaier, K., Golub, T. 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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+extract+cytokines+and+matrix+metalloproteinases+from+Schirmer+strips+and+analyze+using+Luminex.">A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex.</a> Mol Vis. 17, 1056-1063 (2011).</li><li>Liu, J., Kibiki, G., Maro, V., Maro, A., Kumburu, H., Swai, N., Taniuchi, M., Gratz, J., Toney, D., Kang, G., Houpt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+reverse+transcription+PCR+Luminex+assay+for+detection+and+quantitation+of+viral+agents+of+gastroenteritis.">Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis.</a> J. Clin. Virol. 50, 308-313 (2011).</li><li>de Jager, W., Prakken, B. J., Bijlsma, J., WJ Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+multiplex+immunoassay+performance+in+human+plasma+and+synovial+fluid+following+removal+of+interfering+heterophilic+antibodies.">Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.</a> J. Immunol. Methods. 300, 124-135 (2005).</li><li>Codorean, E., Nichita, C., Albulescu, L., Raducan, E., Popescu, I. D., Lonita, A. C., Albulescu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+xMAP+and+ELISA+cytokine+profiles;+development+and+validation+for+immunotoxicological+studies+in+vitro.">Correlation of xMAP and ELISA cytokine profiles; development and validation for immunotoxicological studies in vitro.</a> Roum. Arch. Microbiol. Immunol. 69, 3-19 (2010).</li><li>de Jager, W., te Velthuis, H., Prakken, B. J., Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+15+human+cytokines+in+a+single+sample+of+stimulated+peripheral+blood+mononuclear+cells.">Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.</a> Clin. Diagn. Lab. Immunol. 10, 133-139 (2003).</li><li>DuPont, N. C., Wang, K. H., Wadhwa, P. D., Culhane, J. F., Nelson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+comparison+of+Luminex+multiplex+cytokine+analysis+kits+with+ELISA:+Determinations+of+a+panel+of+nine+cytokines+in+clinical+sample+culture+supernatants.">Validation and comparison of Luminex multiplex cytokine analysis kits with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants.</a> J. Reprod. Immunol. 66, 175-191 (2005).</li><li>Richens, J. L., Urbanowicz, R. A., Metcalf, R., Corne, J., O'Shea, P., Fairclough, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+validation+and+comparison+of+multiplex+cytokine+kits.">Quantitative validation and comparison of multiplex cytokine kits.</a> Journal of Biomolecular Screening. 15, 562-568 (2010).</li><li>Rizzi, G., Zhang, Y. J., Latek, R., Weiner, R., Rhyne, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+development+of+a+Luminex-based+assay+for+the+detection+of+human+IL-23.">Characterization and development of a Luminex-based assay for the detection of human IL-23.</a> Bioanalysis. 2, 1561-1572 (2010).</li></ol>

この作品は、ルミネックス·コーポレーションで製造装置をルミネックス·コーポレーションで行われた。 R＆Dシステム＆EMDミリポアルミネックス·コーポレーションの戦略的パートナーであり、多重XMAPベースのアッセイを開発し、実用化するためにライセンスされています。

ルミネックスXMAPプラットフォームへのELISAアッセイの変換はストレプトアビジンフィコエリスリン（SA-PE）の典型的なELISAキットにストレプトアビジン西洋ワサビペルオキシダーゼ（SA-HRP）を代入すると、パフォーマンスを最適化するような単純なことができます。地面からXMAPイムノアッセイを作成したい人のために、これはまた、抗体ペアの高速、多重評価を可能にする簡単なプロトコルを使用して達成することができます。 XMAPアッセイ用試薬は、簡単にMagPlex低濃度微小球に結合するように指定された捕捉抗体のキットをカップリングXMAP抗体を用いて調製した。より高濃度のマイクロスフェアの同じアッセイの性能を提供しながら、低濃度微小球の使用は、アッセイ開発のコストを削減します。コー​​トにELISAプレートのウェルを必要とし、24時間、治療に続いて - 結合MagPlexミクロスフェアを調製するために必要な時間は、はるかに高速22よりも約3時間であり、コー​​ティングされた井戸の。 XMAPアッセイの性能は、検出限界の点でELISA（&lt;4 pg / mLの対&gt; 31 pg / mLの）とダイナミック·レンジ（8000 pg / mLの対16 &lt;〜4 pg / mLの&gt;に優れているpg / mLの1000〜pg / mLの）。プレートリーダーは、アッセイのダイナミックレンジの上限を制限し、3または4 ODである限られたODの範囲を持っています。 間違いなく、すべての抗体は、ELISAフォーマットで動作し、ELISA法ではうまく動作しないすべての抗体は、XMAPアッセイフォーマットに容易に譲渡することはできません。 XMAPアッセイ（すなわち、同時に実行される）多重化することができますしかし、それは、アッセイに使用するための最良のペアを識別するために、同時に複数の捕捉および検出抗体の組み合わせを評価することが可能である。このプロセスは、一度に1つのペアの評価に限定されているELISAの開発手順に比べてかなりの時間と試薬を節約できます。二つ以上の抗体ペアは等価的に実行する必要があります。アッセイのその他のパペア（たとえば、可用性、コスト等）の適合性を決定するために考慮される。 XMAPアッセイに改良されたアッセイのパフォーマンスと柔軟性に加えて、大幅なコスト削減もあります。 XMAPアッセイの1つのウェルで使用されているビーズのために必要な量は約7.5 ngのであるのに対し、抗体の推奨量は、コート、単一のウェルELISAプレートは400ngであるために必要。 XMAPアッセイで使用されている場合このように、1つのウェルELISAに必要な抗体の量は、50以上のテスト結果を提供します。貴重なサンプルを含むアプリケーションでは、XMAPも大きな利点があります。 XMAPアッセイに必要な量はその半分、または少なくすることができます一方、ELISAのために推奨されるサンプルの量は100μLです。 優れたダイナミックレンジと感度を持つアッセイを製造しながら要約すると、ルミネックスXMAPプラットフォームへのELISAアッセイの変換は、単純な効率的でコスト節約になります。

<table border="1"><tbody><tr><td> 説明 </td><td> ベンダー </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>人間TNF-α/TNFSF1ADuoSet ELISAキット</td><td> R＆Dシステムズ</td><td> DY210 </td><td>モノクローナル抗体およびビオチン結合ポリクローナル抗体、組換えTNF-αタンパク質の標準が含まれています</td></tr><tr><td> TNF-αに対するモノクローナル抗体</td><td>アブカム</td><td> Ab18696 </td><td>抗体、クローンCH8820をキャプチャする</td></tr><tr><td> TNF-αに対するモノクローナル抗体</td><td>アブカム</td><td> Ab16166 </td><td>ビオチン結合の検出抗体、クローンAS1 </td></tr><tr><td> TNFアルファタンパク質</td><td>アブカム</td><td> Ab9642 </td><td>組換えTNF-αタンパク質の標準</td></tr><tr><td> TNF-αに対するモノクローナル抗体</td><td> Novus </td><td> NBP1-50115 </td><td>抗体、クローン4H31をキャ​​プチャする</td></tr><tr><td> TNF-αに対するモノクローナル抗体</td><td>ノーバス</td><td> NB100-78162 </td><td>ビオチン結合の検出抗体、クローンMAb11 </td></tr><tr><td> TNFアルファタンパク質</td><td>ノーバス</td><td> NBC1-18460 </td><td>組換えTNF-αタンパク質の標準</td></tr><tr><td> TNF-αに対するモノクローナル抗体</td><td> EMDミリポア</td><td> MAB1141 </td><td>抗体、クローン3C7.2をキャプチャする</td></tr><tr><td> TNF-αに対するポリクローナル抗体</td><td> EMDミリポア</td><td> 654250 </td><td>ウサギポリクローナル検出抗体</td></tr><tr><td>ストレプトアビジン - フィコエリスリン</td><td>モス</td><td> SAPE-001 </td><td>ルミネックスXMAPアッセイ用蛍光レポーター試薬</td></tr><tr><td> MAGPIX W / Xponentのソフトウェア</td><td>ルミネックス·コーポレーション</td><td> MAGPIX-Xponentの</td><td>ルミネックス·インストゥルメント</td></tr><tr><td> XMAP抗体カップリング（ABC）キット</td><td>ルミネックス·コーポレーション</td><td> 40-50016 </td><td> EDC試薬、スルホ-NHS試薬、活性化バッファー、洗浄バッファー、1.5 mLの反応管と、使い捨てのピペットが含まれています</td></tr><tr><td> MagPlexミクロスフェア、低濃度</td><td>ルミネックス·コーポレーション</td><td> MC10012-ID、MC10013-ID、MC10014-ID、MC10015-ID </td><td>低濃度のビーズ（@ 2.5×10 6ビーズ/ mL）を</td></tr><tr><td> EZ-リンクスルホ-NHS-LC-ビオチン</td><td>サーモフィッシャー</td><td> PI-21335 </td><td>変更されていない検出抗体のビオチン化キット</td></tr><tr><td>テカン無限F200リーダー</td><td>テカン</td><td></td><td> ELISAプレートリーダー</td></tr><tr><td>リン酸緩衝生理食塩水</td><td> Sigma-Aldrich社</td><td> P-3688 </td><td> 1パーセントPBS-BSA、アッセイ緩​​衝液</td></tr><tr><td>ワンパイントコンパクトな超音波洗浄、115 VAC </td><td>コー​​ル·パーマー</td><td> WU-08849から00 </td><td> 55 kHzの効果的な動作周波数を生成する</td></tr><tr><td>磁気チューブセパレータ</td><td>ルミネックス·コーポレーション</td><td> CN-0288から01 </td><td>カップリングの洗浄ステップ内の単一の1.5mlチューブ磁気分離</td></tr><tr><td>磁性板セパレータ</td><td>ルミネックス·コーポレーション</td><td> CN-0269から01 </td><td>アッセイの洗浄ステップで96ウェルプレート磁気分離</td></tr></tbody></table>

conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method

酵素免疫測定法（ELISA）は、長い生命科学の研究と臨床診断の両方の生体試料中の関心のある検体を検出するための主要なツールであった。しかし、ELISA法では限界があります。これは通常、96ウェルマイクロプレートに実行され、井戸は、目的の抗原を捕捉するサンプルの比較的大きな金額を必要とし、捕捉抗体でコーティングされています。井戸の表面積が大きく捕捉抗体の疎水結合には、非特異的結合と増加の背景につながる可能性があります。また、ほとんどのELISA法は、合理的な感度を達成するために、信号の酵素媒介増幅に依存しています。このような増幅は常に線形ではないとすることができますので、スキュー結果。 過去15年間で、新技術は、ELISAの利点を提供している現れではなく、類似したWOより高いスループット、柔軟性、縮小サンプル量、低コストを可能にしましたrkflow 1、2。ルミネックスXMAPテクノロジーは、タンパク質および核酸アプリケーション3から5の両方に適用することができるモノプレックスとマルチプレックスアッセイの両方を可能にするマイクロスフェア（ビーズ）アレイのプラットフォームです。ビーズELISA法に比べて、少ない捕獲抗体と小さなサンプル量を必要とする共有結合より小さい表面積上に固定化された捕捉抗体を持っており、非特異的結合が大幅に削減されます。そのような脳脊髄液、滑液、等6などの制限のサンプルを扱う場合より小さいサンプルボリュームが重要である。検定の多重化は、さらに、単一のサンプルから複数の結果を有効にすると、サンプルボリュームの要件を軽減します。 ルミネックスの最近の改良点は次のとおりです。新しいMAGPIXシステムは、小型で安価な、より使いやすいアナライザ、低濃度の磁気MagPlexミクロス高価なフィルタープレートの必要性を排除し、優れたアッセイ開発に適した作業濃度に来て、低スループットのアプリケーション、およびXMAP抗体カップリング（ABC）キット、プロトコル、試薬、および利息の捕捉抗体ビーズを結合するために必要な消耗品が含まれています。 （キット内容の詳細なリストについては、マテリアルのセクションを参照してください。） この実験では、XMAPプラットフォームにTNF-αサイトカインの事前最適化されたELISAアッセイを変換し、二つの方法7-11の性能を比較します。 TNF-αは自己免疫疾患患者における炎症反応の測定に使用されるバイオマーカーである。 我々は、4つの異なるマイクロセットまたは領域に4つの候補捕捉抗体を結合することによって開始します。一緒に混合したときに、これら4つのセットには、試薬、サンプルと時間を節約し、最高の抗体のペアを決定するための4つの独立した検出抗体を持つすべての4つの候補の同時テストが可能になります。二XMAPアッセイは、2つの最適な抗体のペアとその性能がで構成されている信号の強さ、ダイナミックレンジ、感度に関しては、元のELISAアッセイのそれと比較されます。

Watch this Scientific Journal Video about Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method at JoVE.com

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

ELISAは、簡単にルミネックスXMAPアッセイに変換することができると、多重化の利点を介して、いくつかの抗体は、アッセイのコストを削減しながら、増加した感度とダイナミックレンジの結果、最適な抗体のペアを識別するために同時にスクリーニングすることができる。

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 ...

conversion-capture-elisa-to-luminex-xmap-assay-using-multiplex

Research

JoVE Journal

Biology

43.4K ビュー.  Luminex Corporation. ELISAは、簡単にルミネックスXMAPアッセイに変換することができると、多重化の利点を介して、いくつかの抗体は、アッセイのコストを削減しながら、増加した感度とダイナミックレンジの結果、最適な抗体のペアを識別するために同時にスクリーニングすることができる。

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

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

哺乳動物の組織のレーザーキャプチャーマイクロダイセクション

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

生物学

Cell Capture Using a Microfluidic Device

マイクロ流体デバイスを用いた細胞のキャプチャ

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

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 Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

比色金ナノ粒子アッセイに適用される構造スイッチングアプタマーを選択するための方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

ハイスループットスクリーニングを使用したキナーゼ基質ペアの同定


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

マルチ酵素ハイスループット遺伝的酵素スクリーニングシステムを用いたスクリーニング

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...