이 작품은 Luminex 공사에 의해 재정 지원되었다.

<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. R., Lamb, J. <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+for+high-throughput+gene+expression+signature+analysis.">A method for high-throughput gene expression signature analysis.</a> Genome Biol. 7, 61 (2006).</li><li>VanDerMeid, K. R., Su, S. P., Krenzer, K. L., Ward, K. W., Zhang, J. 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>

이 작품은 Luminex 공사에서 제조 설비를 Luminex 공사에서 이루어졌다. R &amp; D 시스템 및 EMD Millipore는 Luminex Corporation의 전략적 파트너, 멀티 플렉스 xMAP 기반 assays을 개발하고 상용화 허가.

Luminex xMAP 플랫폼으로 엘리사 분석의 변환 streptavidin phycoerythrin (SA-PE)과 전형적인 엘리사 키트에 streptavidin 양고추냉이 퍼옥시데이즈 (SA-HRP)를 대체하고, 성능을 최적화하는 것만 큼이나 간단합니다. 최대 땅속에서 xMAP 면역 분석법을 생성하고자하는 사람들을 위해, 이것은 또한 항체 쌍의 빠른, 멀티 플렉스 평가있게 간단한 프로토콜로 수행할 수 있습니다. xMAP 분석을위한 시약 쉽게 부부 지정 캡처 항체에 MagPlex 낮은 농도 microspheres에 xMAP 항체 커플링 키트를 사용하여 작성되었다. 낮은 농도 microspheres의 사용은 높은 농도 microspheres의 동일한 분석 성능을 제공하면서 분석 개발 비용을 줄여줍니다. 결합 MagPlex microspheres를 준비하는 데 필요한 시간의 양은 어느가 22보다 훨씬 빠른, 약 3 시간 - 24 시간 치료 뒤에는 코트 엘리사 플레이트의 우물을 의무코팅 위. xMAP 분석의 성능을 탐지 (&lt;4 PG / ML 대&gt; 31 PG / ML) 및 동적 범위 (&lt;4 PG / ML로&gt; 8000 PG / ML 대 16 한계의 측면에서도 엘리사에게 우수 PG / ML 1000 PG / ML). 플레이트 리더는 분석을위한 동적 범위의 상한선을 제한, 중 3이나 4 OD는 제한된 OD의 범위가 있습니다. 의심의 여지가없이, 모든 항체는 엘리사 형식으로 작동하며 엘리사에 잘 작동하지 않을 모든 항체는 xMAP 분석 형식으로 쉽게 이전할 수 있습니다. xMAP의 assays가 (동시에 실행 IE) 멀티 플렉스 수 있기 때문에 그러나, 그것은 분석을 위해 사용하는 가장 좋은 쌍을 식별하는 동시에 여러 캡처 및 탐지 항체 조합을 평가하는 것이 가능합니다. 이 과정은 상당한 시간과 한 번에 한 쌍의 평가로 제한됩니다 엘리사 개발 절차와 비교 시약을 저장합니다. 두 개 이상의 항체 쌍은 equivalently 수행되어야하고 분석의 다른 매개 변수 수쌍 (예, 가용성, 비용 등)의 적합성을 결정 간주됩니다. xMAP 분석과 향상된 분석 성능과 유연성뿐만 아니라, 상당한 비용 절감 효과도있다. xMAP 분석을 잘 하나에 사용되는 구슬에 필요한 수량이 약 7.5 NG이지만 항체의 권장 수량은 코트 한 우물 엘리사 플레이트가 400ng이다에 필요합니다. 따라서, 하나 엘리사에 필요한 항체의 양을 잘 xMAP 분석에 사용되는 경우, 50 개 이상의 테스트 결과를 제공합니다. 귀중한 샘플을 포함한 어플 리케이션의 경우, xMAP 또한 상당한 장점이 있습니다. xMAP 분석에 필요한 볼륨 그 중 절반 이하가 될 수 반면 엘리사 권장 시료의 부피는 100 μL입니다. 우수한 동적 범위와 감도와 분석을 생산하면서 요약에 Luminex xMAP 플랫폼으로 엘리사 분석의 변환은, 단순한 효율 및 비용 절감이다.

<table border="1"><tbody><tr><td> 설명 </td><td> 공급 업체 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 인간 TNF-α/TNFSF1A DuoSet 엘리사 키트 </td><td> R &amp; D 시스템 </td><td> DY210 </td><td> 단클론 및 비오틴 결합 polyclonal 항체 및 재조합 TNF-α 단백질 표준을 포함 </td></tr><tr><td> TNF-α에 대한 단클론 항체 </td><td> Abcam </td><td> Ab18696 </td><td> , 클론 CH8820를 항체를 캡처 </td></tr><tr><td> TNF-α에 대한 단클론 항체 </td><td> Abcam </td><td> Ab16166 </td><td> 비오틴 결합 감지 항체, 클론 AS1 </td></tr><tr><td> TNF 알파 단백질 </td><td> Abcam </td><td> Ab9642 </td><td> 재조합 TNF-α 단백질 표준 </td></tr><tr><td> TNF-α에 대한 단클론 항체 </td><td> Novus </td><td> NBP1-50115 </td><td> , 클론 4H31을 항체를 캡처 </td></tr><tr><td> TNF-α에 대한 단클론 항체 </td><td> Novus </td><td> NB100-78162 </td><td> 비오틴 결합 감지 항체, 클론 MAb11 </td></tr><tr><td> TNF 알파 단백질 </td><td> Novus </td><td> NBC1-18460 </td><td> 재조합 TNF-α 단백질 표준 </td></tr><tr><td> TNF-α에 대한 단클론 항체 </td><td> EMD Millipore </td><td> MAB1141 </td><td> 항체를 캡처, 3C7.2을 복제 </td></tr><tr><td> TNF-α에 대한 Polyclonal 항체 </td><td> EMD Millipore </td><td> 654,250 </td><td> 토끼 polyclonal 감지 항체 </td></tr><tr><td> Streptavidin-Phycoerythrin </td><td> 이끼 </td><td> SAPE-001 </td><td> Luminex xMAP 분석을위한 형광 기자 시약 </td></tr><tr><td> MAGPIX W / xPONENT 소프트웨어 </td><td> Luminex 공사 </td><td> 석사GPIX-XPONENT </td><td> Luminex의 악기 </td></tr><tr><td> xMAP 항체 커플링 (AB​​C) 키트 </td><td> Luminex 공사 </td><td> 40-50016 </td><td> EDC 시약, Sulfo-보건국 시약, 활성화 버퍼, 세차 버퍼, 1.5 ML 반응 튜브, 그리고 일회용 pipettes 포함 </td></tr><tr><td> MagPlex Microspheres, 낮은 농도 </td><td> Luminex 공사 </td><td> MC10012-ID, MC10013-ID, MC10014-ID, MC10015-ID </td><td> 낮은 농도 구슬 (@ 2.5 X 10 6 비드 / ML) </td></tr><tr><td> EZ-링크 Sulfo-NHS-LC-비오틴 </td><td> 써모 피셔 </td><td> PI-21335 </td><td> 수정되지 않은 항체 검출을위한 Biotinylation 키트 </td></tr><tr><td> Tecan 무한 F200 리더 </td><td> Tecan </td><td></td><td> 엘리사 플레이트 리더 </td></tr><tr><td> 인산염은 염분 버퍼 </td><td> 시그마 - 올드 리치 </td><td> P-3688 </td><td> 1퍼센트 PBS-BSA, 분석 완충액 </td></tr><tr><td> 한 파인트 소형 초음파 청소기, VAC 115 </td><td> 콜 - Parmer </td><td> WU-08849-00 </td><td> 55 kHz에서의 효과적인 운영 주파수를 제작 </td></tr><tr><td> 마그네틱 튜브 구분 기호 </td><td> Luminex 공사 </td><td> CN-0288-01 </td><td> 커플링 세탁 단계에서 단일 1.5mL 튜브 자성 분리를 위해 </td></tr><tr><td> 마그네틱 플레이트 구분 기호 </td><td> Luminex 공사 </td><td> CN-0269-01 </td><td> 검정 세척 단계에서 96 - 웰 플레이트 자성 분리를 위해 </td></tr></tbody></table>

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

효소 결합 면역 분석 (엘리사)는 오랫동안 생명 과학 연구 및 임상 진단 모두에 대한 생물 학적 시료에 대한 관심 analytes의 검출하기위한 중요한 도구가되었습니다. 그러나 엘리사는 제한 사항이 있습니다. 그것은 일반적으로 96 - 웰 microplate에서 수행되며, 우물은 관심 항원을 포착하기 위해 시료의 비교적 큰 금액을 요구, 캡처 항체로 코팅됩니다. 우물의 넓은 표면적과 캡쳐 항체의 소수성 바인딩도 아닌 특정 바인딩과 증가 배경으로 이어질 수 있습니다. 또한 대부분의 ELISAs는 합리적인 감도를 달성하기 위해서는 신호의 효소 - 매개 증폭에 의존. 이러한 증폭은 항상 선형 아니며 수 있으므로 스큐 결과입니다. 지난 15 년 동안 새로운 기술은 비슷한 우와 함께하는가 엘리사의 혜택을 제공뿐만 아니라, 높은 처리량, 향상된 유연성, 감소 샘플 볼륨, 그리고 낮은 비용을 가능하게 떠오르고rkflow 1, 2. Luminex xMAP 기술 monoplex과 단백질과 핵산 응용 3-5 모두에 적용할 수있는 멀티 플렉스 assays를 모두 가능하게 microsphere (구슬) 배열 플랫폼입니다. 구슬은 엘리사에 비해 덜 캡처 항체와 작은 샘플 볼륨을 필요로하는, covalently 작은 표면적에 고정화 캡처 항체를 가지고 있고, 이외의 구체적인 구속력이 크게 줄어 듭니다. 같은 뇌척수, 활액 액체 등 6 등 제한 샘플 작업시 작은 샘플 볼륨이 중요하다. 분석을 다중화하는 것은 더 이상 하나의 샘플에서 여러 결과를함으로써, 샘플 볼륨 요구 사항을 줄여줍니다. Luminex에 의한 최근의 향상된 기능은 다음과 같습니다 : 새로운 MAGPIX 시스템, 작고, 저렴, 쉽게 - 사용하기 분석기, 낮은 농도 마그네틱 MagPlex Microspheres 고가의 필터 플레이트에 대한 필요성을 제거하고 더 나은 분석 개발에 적합 근무 농도에 와서낮은 처리량 어플 리케이션 및 xMAP 항체 커플링 (AB​​C) 키트, 프로토콜, 시약 및 관심 캡처 항체에 구슬을 커플링에 필요한 소모품을 포함합니다. (키트 내용의 자세한 목록은 자료 섹션을 참조하십시오.) 본 실험에서는 xMAP 플랫폼 TNF-알파 시토킨위한 사전 최적화된 엘리사 분석을 변환하고 두 가지 방법 7-11의 성능을 비교합니다. TNF-알파는 면역 장애 환자에서 염증 반응의 측정에 사용되는 biomarker입니다. 우리는 네 가지 microsphere 세트 또는 지역 네 가지 후보 캡처 항체를 결합하여 시작합니다. 함께 혼합하면 이들 4 세트는 최고의 항체 쌍, 저장 시약, 샘플 및 시간을 결정하는 4 개의 별도의 탐지 항체를 가진 네 명의 후보 중 동시 테스트하실 수 있습니다. 두 xMAP의 assays 그런 다음 두 가장 최적 항체 쌍과 건설과 그 성과가있다신호 강도, 동적 범위 및 감도에 관해서 원래 엘리사 분석의 비교.

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

엘리사 쉽게 Luminex xMAP 분석으로 변환될 수 있으며, 멀티플렉싱, 여러 항체의 장점을 통해 분석 비용을 줄이는 한편, 증가 감도 및 다이나믹 레인지 결과, 최적 항체 쌍을 식별하는 동시에 상영 할 수 있습니다.

멀티 플렉스 항체 검사 방법을 사용하여 Luminex xMAP 분석에 캡처 엘리사의 전환

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

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43.4K 조회수.  Luminex Corporation. 엘리사 쉽게 Luminex xMAP 분석으로 변환될 수 있으며, 멀티플렉싱, 여러 항체의 장점을 통해 분석 비용을 줄이는 한편, 증가 감도 및 다이나믹 레인지 결과, 최적 항체 쌍을 식별하는 동시에 상영 할 수 있습니다.

비디오: 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.

포유 동물 조직의 레이저 캡처 Microdissection

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

연구

생물학

Cell Capture Using a Microfluidic Device

Microfluidic 장치를 사용하여 셀 캡처

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.

칙의 전체 마운트 항체 스테 이닝을위한 방법

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.

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans</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 채널의 변조기를 식별을위한 형광 검사 분석

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.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

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.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

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.

색채 금 나노 입자 분석에 적용 구조 전환 앱 타머를 선택하는 방법

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.

높은 처리량 검열을 사용 키나제 기판 쌍의 식별


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).

멀티 효소 높은 처리량 유전자 효소 검사 시스템을 사용하여 상영

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