이 작품은 건강 보조금 1R21NS073097-01과 1R01DK082884 (JSD)과 국립 연구소 부여 PIER11VCTR에 대한 재단의 국립 연구소에서 자금에 의해 지원되었다.

<ol>
	<li>Ehrlich, 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=Inward+rectifier+potassium+currents+as+a+target+for+atrial+fibrillation+therapy.">Inward rectifier potassium currents as a target for atrial fibrillation therapy.</a> J. Cardiovasc. Pharmacol. 52 (2), 129 (2008).</li><li>Bhave, G., Lonergan, D., Chauder, B. A., Denton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+modulators+of+inward+rectifier+K++channels:+recent+advances+and+future+possibilities.">Small-molecule modulators of inward rectifier K+ channels: recent advances and future possibilities.</a> Future Med. Chem. 2 (5), 757 (2010).</li><li>Swartz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarantula+toxins+interacting+with+voltage+sensors+in+potassium+channels.">Tarantula toxins interacting with voltage sensors in potassium channels.</a> Toxicon. 49 (2), 213 (2007).</li><li>Jin, W., Lu, 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+novel+high-affinity+inhibitor+for+inward-rectifier+K++channels.">A novel high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 37 (38), 13291 (1998).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+stable+form+of+tertiapin:+a+high-affinity+inhibitor+for+inward-rectifier+K++channels.">Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 38 (43), 14286 (1999).</li><li>Roy, A., McDonald, P. R., Sittampalam, S., Chaguturu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+high+throughput+drug+discovery+in+the+public+domain:+a+Mount+Everest+in+the+making.">Open access high throughput drug discovery in the public domain: a Mount Everest in the making.</a> Curr. Pharm. Biotechnol. 11 (7), 764 (2010).</li><li>Weaver, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thallium-sensitive,+fluorescence-based+assay+for+detecting+and+characterizing+potassium+channel+modulators+in+mammalian+cells.">A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.</a> J. Biomol. Screen. 9 (8), 671 (2004).</li><li>Lewis, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+reveals+a+small-molecule+inhibitor+of+the+renal+outer+medullary+potassium+channel+and.">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and.</a> 76 (5), 1094 (2009).</li><li>Bhave, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+selective+small-molecule+inhibitor+of+Kir1.1,+the+renal+outer+medullary+potassium+channel.">Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.</a> Mol. Pharmacol. 79 (1), 42 (2011).</li><li>Raphemot, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterization,+and+structure-activity+relationships+of+an+inhibitor+of+inward+rectifier+potassium+(Kir)+channels+with+preference+for+Kir2.3,+Kir3.x,+and+Kir7.1.">Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1.</a> Front Pharmacol. 2, 75 (2012).</li><li>Niswender, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+of+Gi/o-linked+G+protein-coupled+receptor+coupling+to+potassium+channels+provides+new+insights+into+the+pharmacology+of+the+group+III+metabotropic+glutamate+receptors.">A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors.</a> Mol. Pharmacol. 73 (4), 1213 (2008).</li></ol>

관심 없음 충돌이 선언 없습니다.

데이터 치료 : 일단 데이터가 분석에 일반적인 단계는 실험, F 0 초에 초기 값으로 각도의 형광 반응, F를 정규화, 포함한다 수집하고 있습니다. 이것은 일반적으로 &quot;정적 비율&quot;로 지칭하고 &quot;F / F 0&quot;상징합니다. F 0 표시기 염료에 의해 ​​지배되는 경우에는 정적 비율 작업이 실질적으로 같은 조명, 신호 컬렉션에 disuniformities 등 여러 요인...

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.
The formula in step 7.1 of the Protocol has been updated from:
Z prime = 1- (3SDp + 3SDn)/|meanp + meann |
to:
Z prime = 1- (3SDp + 3SDn)/|meanp - meann |

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.

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

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> pcDNA5/TO </td><td> Invitrogen </td><td> V1033-20 </td><td> 테트라 사이클린-inducible 표현 벡터 </td></tr><tr><td> T-렉스 - HEK293 세포 </td><td> Invitrogen </td><td> R71007 </td><td> 테트라 사이클린-inducible 세포 라인 </td></tr><tr><td> Lipofectamine LTX / 플러스 시약 </td><td> Invitrogen </td><td> 15338100 </td><td> Transfection 시약 </td></tr><tr><td> FBS </td><td> 애틀랜타 체액 </td><td> S11550 </td><td> 세포 배양 미디어 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11,965 </td><td> 세포 배양 미디어 </td></tr><tr><td> Hygromycin B </td><td> Invitrogen </td><td> 10687-010 </td><td> 세포 배양 미디어 </td></tr><tr><td> Blasticidin S </td><td> Invitrogen </td&gt; <td> R210-01 </td><td> 세포 배양 미디어 </td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Invitrogen </td><td> 15,140 </td><td> 세포 배양 미디어 </td></tr><tr><td> 무료 HBSS - 이가의 </td><td> Mediatech </td><td> 21022CV </td><td> 셀 세척 </td></tr><tr><td> 트립신 - 0.25 % </td><td> Mediatech </td><td> 25053CI </td><td> 세포 분리 </td></tr><tr><td> 테트라 사이클린-HCL </td><td> 시그마 </td><td> T9823 </td><td> 유도 시약 </td></tr><tr><td> Dialyzed FBS </td><td> 애틀랜타 체액 </td><td> S12650 </td><td> 도금 미디어 </td></tr><tr><td> FluoZin-2 </td><td> Invitrogen </td><td> F24189 </td><td> 형광 염료 </td></tr><tr><td> Pluronic F-127 </td><td> Invitrogen </td><td> P-3000MP </td><td> 염료로드 </td></tr><tr><td> HBSS </td><td> Invitrogen </td><td> 14,175 </td><td> 검정 버퍼 </td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15,630 </td><td> 검정 버퍼 </td></tr><tr><td> NaHCO 3 </td><td> 시그마 </td><td> S6297 </td><td> TL + 자극 버퍼 </td></tr><tr><td> MgSO 4 </td><td> 시그마 </td><td> M2643 </td><td> TL + 자극 버퍼 </td></tr><tr><td> 카소 4 • 2H 2 O </td><td> 시그마 </td><td> C3771 </td><td> TL + 자극 버퍼 </td></tr><tr><td> D-포도당 </td><td> 시그마 </td><td> G7528 </td><td> TL + 자극 버퍼 </td></tr><tr><td> 탈륨 황산 </td><td> 올드리치 </td><td> 204625 </td><td> TL + 자극 버퍼 </td></tr><tr><td> HEPES </td><td> 시그마 </td><td> H4034 </td><td> TL + 자극 버퍼 </td></tr><tr><td> DMSO </td><td> 시그마 </td><td> D4540 </td><td> 용제 </td></tr><tr><td> 여덟 채널 전자 pipettor </td><td> Biohit </td><td> E300 </td><td> 384에서 셀 도금 -잘 접시 </td></tr><tr><td> BD PureCoat 아민 - 코팅 384 잘 접시 </td><td> BD Biosciences </td><td> 356719 </td><td> 검정 microplates </td></tr><tr><td> 에코는 384 자 폴리 프로필렌 microplate (384PP)를 자격 </td><td> Labcyte </td><td> P-05525 </td><td> 합성 소스 microplates </td></tr><tr><td> 384도 폴리 프로필렌 microplates </td><td> Greiner 바이오 하나 </td><td> 781280 </td><td></td></tr><tr><td> Multidrop 콤비 시약 디스펜서 </td><td> 열 과학 </td><td> 5840300 </td><td></td></tr><tr><td> ELx405 microplate 와셔 </td><td> BioTek </td><td> ELx405HT </td><td> 자동 셀 세척 </td></tr><tr><td> 액체 처리기를 반향 </td><td> Labcyte </td><td> Labcyte 에코 550 </td><td></td></tr><tr><td> 브라보, 자동화 된 액체 처리 플랫폼 </td><td> 애질런트 테크놀로지스 </td><td> 표준 모델 </td><td></td></tr><tr><td> Hamama츠 FDSS 6000 </td><td> 하마 마츠 </td><td></td><td> 운동 이미징 플레이트 판독기 </td></tr></tbody></table> 표 1. 재료 및 시약의 목록입니다.

high-throughput screening for small-molecule modulators of inward rectifier potassium channels

안쪽 정류기의 칼륨 (Kir) 채널 가족의 특정 구성원은 고혈압, 심방 세동과 통증 1,2 등의 질환의 다양한에 대한 약물 목표를 postulated 있습니다. 대부분의 경우, 그러나, 자신의 치료 가능성이나 기본적인 생리 기능을 이해하는 방향으로 진행 좋은 약리 도구의 부족에 의해 느려졌습니다. 사실, 안쪽 정류기 제품군의 분자 약리학 훨씬 nanomolar 화성과 매우 선택적 펩타이드 독소의 변조기의 번호가 3 발견되었습니다 된 전압 개폐 칼륨 (KV) 채널의 S4 superfamily의 뒤에 느껴지있다. 벌 독이 독소의 tertiapin 및 파생 4,5 Kir1.1와 Kir3 채널의 강력한 억제제하지만, 펩티드는 실험적으로 therapeutically뿐만 아니라 제한된 용도의 antigenic 속성과 가난한 생체 이용률, 신진 대사 안정성과 조직 penetrance로 인해 수 있습니다. 강력한 개발향상된 약리 특성을 가진 선택적 작은 분자 프로브는 완전히 생리학과 Kir 채널 치료 가능성을 이해하는 열쇠가 될 것이다. 학술 과학자들은 더 나은 약리학 6 필요로하는 분자 표적 및 신호 전달 경로에 대한 프로브 발견 캠페인을 시작하기 위해 국립 보건원에서 지원하는 분자 도서관 프로브 제작 센터 네트워크 (MLPCN (주) NIH) 공통 기금은 기회를 만들었습니다. MLPCN는 연구자에게 산업 규모 검진 센터, 의약 화학, 정보학은 유전자와 유전자 네트워크의 기능을 명료하게하다하는 작은 분자 프로브를 개발 지원에 대한 액세스를 제공합니다. MLPCN에 항목을 얻을의 중요한 단계는 높은 처리량 검사 (HTS)에 대한 의무입니다 강력한 대상 또는 경로 별 분석의 개발입니다. 여기, 우리는 형광 기반의 탈륨 (TL +) 자속 assa을 개발하는 방법에 대해 설명합니다높은 처리량 화합물 심사 7,8,9,10에 대한 Kir 채널 기능의 y는. 분석은 K의 투자율 + K + congener TL +에 채널 기공을 기반으로합니다. 상업적으로 이용 가능한 형광 TL + 기자 염료가 모공을 통해 TL의 transmembrane 유량을 + 감지하는 데 사용됩니다. BTC, FluoZin-2 및 FluxOR 7,8 : TL + 자속 assays에 적합 적어도 세 상업적으로 이용 가능한 염료가 있습니다. 이 프로토콜은 FluoZin-2를 사용하여 분석 개발에 대해 설명합니다. 원래 개발 및 아연 표시로 판매했지만, FluoZin-2 전시 TL시 형광 방출 + 바인딩에 강력하고 복용 종속적 인 증가합니다. 우리는 FluxOR는 7,8 사용할 수 등 9,10 할 계속하기 전에 FluoZin-2와 협력하기 시작했습니다. 그러나, 분석 개발 단계는 세 염료에 본질적으로 동일하며, 사용자는 자신의 특정 N에 가장 적합한 어떤 염료 확인해야합니다eeds. 우리는 또한 MLPCN에 항목으로 간주 할 수에 도달해야합니다 검정의 성능 벤치 마크에 대해 설명합니다. TL +는 쉽게 대부분의 K + 채널을 permeates 때문에, 분석은 대부분의 K + 채널 대상에 적용해야합니다.

테트라 사이클린-inducible 표현 시스템의 사용은 내생 경로와 관심 Kir 채널을 통해 TL + 자속을 구별하기에 편리한 내부 통제를 제공합니다. 그림 1 쇼 실험의 다른 유형에 사용되는 셀 도금지도의 예. uninduced 또는 테트라 사이클린 - 유도 세포를 포함하는 우물의 위치는 서로 다른 색상으로 표시됩니다. 그림 2A는 분석 개발과 화합물 심사에 대한 최적의 TL + ...

Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

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

안쪽 정류기의 칼륨 (Kir) 높은 처리량 화합물 심사에 대한 채널의 활동을 측정하기위한 양적 형광 분석을 개발하고 검증하기위한 방법이 제공됩니다.

안쪽 정류기의 칼륨 채널의 소형 분자 변조기에 대한 높은 처리량 검사

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

high-throughput-screening-for-small-molecule-modulators-inward

Research

JoVE Journal

Biology

14.9K 조회수.  Vanderbilt University School of Medicine. 안쪽 정류기의 칼륨 (Kir) 높은 처리량 화합물 심사에 대한 채널의 활동을 측정하기위한 양적 형광 분석을 개발하고 검증하기위한 방법이 제공됩니다.

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

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.

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스

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

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생물학

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

에서 곰팡이 Endoglucanase 활동의 높은 처리량 검사 대장균</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

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 ...

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 of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

의 Permeabilization의 방법<em&gt; 초파리</em작은 분자 활동의 분석 실험을위한&gt; 태아

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

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

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 ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.