이 작품은 미국 공중 보건 서비스 상을 NS-071530에 의해 지원되었다.

1. 세포의 준비 <ol><li> 5% CO 2의 humidified 분위기에서 10 %의 목마 혈청 및 37 ° C에서 문화를 유지와 보완 Dulbecco의 수정된 이글 배지 (DMEM)에서 뇌하수체 AtT20 세포를 성장. </li><li> 표준 trypsinization 절차를 사용하여 세포에게 5-7일마다 subculturing하여 문화를 유지합니다. </li><li> 검정, 투명 하단의 문장은 우물, 폴리-L-라이신의 50 μL와 96 - 웰 플레이트. 우물은 인큐베이터에서 30 분 동안 건조하도록 허용합니다. </li><li> 물론 당 30,000의 세포 밀도에서와 3-4일 동안 배양기에 세포를 저장하는 200 μL 미디어를 포함하는 96 - 웰 플레이트에 플레이트 세포를. </li><li> 천천히 세포 단일층에서 문화 매체를 기음 한 끝에 진공에 연결된 마개 4리터 항아리와 다른 끝에 200 μL 피펫 팁으로 구성된 열망 설정을 사용합니다. </li><li> 정상적인 완충 용액 200 μL (132 MM NaCl, 5 KCl MM, 1m로 세포를 씻어M CaCl 2, 1 밀리미터 MgCl 2, 5 밀리미터 덱스 트로 오스, 5 밀리미터 HEPES, NaOH로 pH를 7.4). </li><li> 각도에 막 잠재적인 감광 염료 (MPD) DiBAC 4 (3) 또는 HLB 021-152 (5 μm의)를 포함하는 완충 용액 200 μL를 추가하고 37에서 40 분 대한 세포를 품어 ° C. </li></ol> 2. 시험 화합물과 세포 치료의 작성 <ol><li> Versette (ThermoFisher) 자동 액체 처리 시스템을 사용하여 화합물 라이브러리에서 테스트 화합물의 시리얼 희석 (DMSO의 주식 = 10 밀리) (96 - 웰 형식) 준비합니다. </li><li> , 96 - 웰 플레이트를 제거 AtT20 세포를 포함, 인큐베이터에서와 플레이트는 실내 온도로 돌아갈 수 있습니다. 오래된 버퍼를 기음과 세포에 MPD를 포함하는 새로운 버퍼 솔루션을 적용합니다. </li><li> 액체 처리 시스템은 다양한 시험 화합물의 농도 (10 NM 10 μm의까지) (2-20 μL) 및 제어 솔루션 (0.1 % DMSO)을 (버퍼 용액 containi에 적용 사용세포를 포함하는 96 - 웰 플레이트에 NG MPD). </li><li> 형광 측정에 대한 사전 테스트 화합물로 약 5 분 동안 세포를 품어. </li></ol> 3. GIRK 채널의 활성화, 형광 측정 및 분석 <ol><li> Biotek Synergy2 형광 플레이트 판독기에 96 - 웰 플레이트를 삽입하고 각각 여기 및 방출 520의 파장과 560 nm의에서 형광 배경 신호를 구하십시오. </li><li> Synergy2 인젝터 시스템을 사용 GIRK 채널을 활성화 (총 부피 = 220 μL)을 우물에 GPCR의 리간드 (somatostatin = 200 nm의 또는 carbachol이 = 10 μm의) 또는 제어 솔루션 (버퍼 혼자) (20 μL)을 적용합니다. 각각 여기 및 방출 520의 파장과 560 nm의에서 300의 샘플링 기간 동안 10의 간격으로 데이터 포인트를 수집합니다. </li><li> 시간 코스와 Biotek Gen5 소프트웨어를 사용하여 형광 변화의 피크 진폭을 확보하고 추가 분석을 위해 Excel로 데이터를 내보냅니다. </lI&gt; <li> 분석의 신뢰성을 결정하는 Z&#39; 팩터를 계산합니다. Z&#39; 팩터 결정에 사용되는 플레이트는 2 행 긍정의 각 (GPCR의 리간드)과 부정 (버퍼 혼자) 컨트롤이 포함되어 있습니다. Z&#39; 팩터과 같이 정의됩니다 : Z &#39;= 1 - (3σ P + 3σ N) / | μ의 P - μ N | μ P &amp; μ N은 긍정적인 제어 및 부정적인 제어 신호의 수단입니다, 그리고 σ P는 &amp; σ N 긍정적인 컨트롤의 표준 편차와 부정적인 제어 신호를 각각 6 있습니다. 0.5-1.0의 범위에서 Z&#39; - 요인 분석의 품질 6 우수한 것으로 나타났습니다. 0.5 아래 Z&#39;-요인과 플레이트는 분석에서 제외됩니다. </li><li> 형광 신호를 변조 화합물은 내향 정류기 K + 채널 특이성을 확인하는 바 2의 존재 + 또는 tertiapin-Q에 retested됩니다. </li></ol> 4. Representative 결과 GIRK 채널 분석을 사용하여 측정된 형광 신호의 예제는 그림 2에 표시됩니다. AtT20 세포 GPCR의 리간드 somatostatin (200 NM)의 추가는 HLB 021-152 형광 신호의 빠른 시간 종속적인 감소 (그림 2) 발생했습니다. 대조적으로, 제어 솔루션의 첨가 시간 (그림 2) 기준선에 반환되는 형광의 작은 순간적인 상승을 생산했다. 제어 및 somatostatin 솔루션으로 주입 96 - 웰 플레이트에서 최대 형광 값의 비교는 0.7에 0.5의 범위에서 Z&#39;-요인을 주었다. 자세한 부량 들어, 제어 레코드는 somatostatin 레코드에서 빼는되었고 결과 somatostatin에 민감한 신호는 (그림 2) 분석했다. 분석은 신속하게 GIRK 채널 조절 약물을 식별하는 방법을 제공합니다. 예를 들어, 마약 GIRK 차를 억제 그nnel는 세포에서 K + 유출을 방지하여 형광의 GPCR 리간드 - 중재 감소을 감소해야합니다. 차단 GIRK 채널 7, somatostatin-매개 형광 변화의 억제를 생산하는 그림 3, tertiapin-Q, 독소와 세포 치료에 나와 있습니다. Propafenone, 심장 8 블록 GIRK 채널도 형광 변화 (그림 4)를 저해하는 안티 arrhythmic 약물. 분석은 또한 GIRK 채널의 activators을 식별하는 데 유용할 수 있습니다. 용액 (P&gt; 0.5)을 제어할 비해 그러나 에탄올의 응용 (100 &amp; 200 MM)는 GIRK 채널 2,3의 활성은 형광 신호의 큰 변화도 원인이 없습니다. <img src="/files/ftp_upload/3850/3850fig1.jpg" alt="그림 1"/> 그림 1. GIRK 채널 형광 분석의 실험적 디자인. AtT20 세포 membran를 포함하는 버퍼에 incubated 있습니다전자 가능성에 민감한 형광 염료 (D). 염료 분자 세포에 입력하고 세포 내 단백질 (맨 위 패널)에 바인딩시 형광된다. 은 GPCR로 (Som) somatostatin의 바인딩 GIRK 채널 (하단 패널)의 활성화를 일으키는 G 억제 단백질을 (G I) 자극. K는 세포 밖으로 +의 후속 유출은 세포를 종료 것이 더 부정적와 염료 분자가 될 멤브레인 잠재됩니다. 따라서 형광 신호 감소 (하단 패널). <img src="/files/ftp_upload/3850/3850fig2.jpg" alt="그림 2"/> somatostatin 또는 제어 솔루션뿐만 동안 AtT20 세포에서 시간이 지남에 따라 측정 그림 2. 대표 HLB 021-152 형광 신호. 형광 강도 (F / F O)의 비율 (F O) 광고를하기 전에 측정 기준 신호에 의한 somatostatin의 존재 (F) (또는 제어 솔루션)의 신호를 나누어 계산somatostatin의 dition (또는 제어 솔루션). 각 포인트는 평균 ± SE 5-6 우물에서 얻어진을 나타냅니다. Somatostatin 또는 제어 솔루션은 시간이 영 (↓)에 추가되었습니다. 제어 솔루션의 추가는 형광 신호의 작은 과도 증가를 일으켰습니다. 이것은 솔루션의 주입에 의한 온도 변화에서 발생했습니다. <img src="/files/ftp_upload/3850/3850fig3.jpg" alt="그림 3"/> 그림 3. tertiapin-Q (500 NM)와 AtT20 세포 치료로 묶는 방식과 GIRK 채널을 차단하여 형광 신호의 somatostatin-매개 감소를 억제. 각 포인트는 평균 ± SE 4-6 우물에서 얻어진을 나타냅니다. Somatostatin 시간이 영 (↓)에 추가되었습니다. <img src="/files/ftp_upload/3850/3850fig4.jpg" alt="그림 4"/> 그림 4. propafenone (20 μm의)와 AtT20 세포의 치료도에서 somatostatin-매개 감소를 억제형광 신호. 각 포인트는 평균 ± SE 5-6 우물에서 얻어진을 나타냅니다. Somatostatin 시간이 영 (↓)에 추가되었습니다.

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관심의 어떠한 충돌 선언 없습니다.

막 잠재력에 민감한 형광 염료가 이온 채널 조절 약물 9,10을 식별하는 데 사용되었습니다 있지만, 이것의 연결을 GIRK 채널 약물 발견에 대한 자신의 응용 프로그램의 첫 번째 보고서이다. 여기서 제시 GIRK 채널 형광 분석은 리간드 게이트 K + 채널의 심사를위한 빠르고, 신뢰할 수있는 실시간 방식을 제공합니다. 검정은 외인성 재조합 GIRK 채널 11 clonal 셀 라인 (AtT20, PC-12) 내...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> CellGro </td><td> 10-013 </td></tr><tr><td> 말 혈청 </td><td> Invitrogen </td><td> 16050-114 </td></tr><tr><td> 96 - 웰 플레이트 </td><td> 코닝 </td><td> 3603 </td></tr><tr><td> 폴리-L-라이신 </td><td> 시그마 - 올드 리치 </td><td> P4707 </td></tr><tr><td> Somatostatin </td><td> 시그마 - 올드 리치 </td><td> S9129 </td></tr><tr><td> Carbachol </td><td> 시그마 - 올드 리치 </td><td> C4382 </td></tr><tr><td> HLB 021-152 </td><td> AnaSpec </td><td> 89,300 </td></tr><tr><td> Versette 자동 액체 처리기 </td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> Synergy2 형광 플레이트 리더 </td><td> Biotek </td><td></td></tr><tr><td> Gen5 분석의oftware </td><td> Biotek </td><td></td></tr></tbody></table> 표 1. 특정 시약 및 장비의 표.

a fluorescent screening assay for identifying modulators of girk channels

G 단백질 문이 안쪽 정류기 K + (GIRK) 채널은 호르몬과 신경 전달 물질의 광범위한 셀룰러 중재자 역할과 뇌, 심장, 골격근 및 내분비 조직 1,2로 표현됩니다. GIRK 채널들은 플라즈마 멤브레인 바인딩된, G 단백질 결합 수용체 (GPCRs)에 (신경 전달 물질, 호르몬, 약물 등) 리간드의 바인딩에 따라 활성화된다. 이 바인딩은 이후에 바인딩하고 GIRK 채널을 활성화 G 단백질 (G i와 G O)의 자극을 초래합니다. 일단 GIRK 채널은 K의 움직임 +에서 더 많은 부정이 될 가능성이 휴식 멤브레인를 일으키는 세포의 수 있습니다 열었습니다. 결과적으로 뉴런에 GIRK 채널 인증은 자발적인 행동 잠재력의 형성을 감소하고 흥분성의 신경 전달 물질의 석방을 억제. 마음에 GIRK 채널의 활성화함으로써 심장 박동을 둔화 맥박 조정기의 활동을 억제. <p클래스 = &quot;jove_content&quot;&gt; GIRK 채널은 치료 neuropathic 고통, 마약 중독, 심장 arrhythmias 및 기타 장애 3에 대한 새로운 치료 대리인의 개발을위한 소설 대상을 나타냅니다. 그러나 이러한 채널 약리학이 크게 미개척 남아 있습니다. 안티 arrhythmic 대리인, 치료약의 약물과 항우울제를 포함한 약물의 숫자 GIRK 채널을 차단 있지만, 이러한 억제는 선택 아니므로 상대적으로 높은 약물 농도 3 시에 발생합니다. 여기서는 GIRK 채널의 새로운 변조기를 식별을위한 실시간 검사 분석을 설명합니다. 본 분석에서는 GIRK 채널을 표현의 연결을 AtT20 세포는 같은 비스 - (1,3-dibutylbarbituric 산)와 같은 멤브레인 가능성에 민감한 형광 염료와 함께로드됩니다 trimethine oxonol [DiBAC 4 (3)] 또는 HLB 021-152 (그림 1 ). 염료 분자 세포 (그림 1)에 강력하게 형광등 아래의 이해가된다. 치료GPCR의 리간드와 세포 열 GIRK 채널 자극. 세포의 결과 K + 유출 아웃 더 음수가 될 수있는 멤브레인 잠재력과 감소하는 형광 신호 (그림 1)을 일으 킵니다. 따라서 GIRK 채널을 통해 K + 유출을 조절 약물은 형광 플레이트 리더를 사용 assayed 수 있습니다. 다른 이온 채널 심사 assays 같은 원자 흡수 분광법 4 radiotracer 분석 5와는 달리, GIRK 채널 형광 분석은 빠른 실시간 저렴 심사 절차를 제공합니다.

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

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G 단백질 문이 안쪽 정류기 K와 상호 작용하는 약물을 식별을위한 실시간 검사 절차<sup&gt; +</sup&gt; (GIRK) 채널이 설명되어 있습니다. 검정은 GIRK 채널 활동을 측정하는 막 잠재력에 민감한 형광 염료를 활용합니다. 이 기술은 세포 라인의 숫자에 사용하기위한 적응이다.

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

Research

JoVE Journal

Biology

14.3K 조회수.  University of South Carolina, School of Medicine. G 단백질 문이 안쪽 정류기 K와 상호 작용하는 약물을 식별을위한 실시간 검사 절차 + (GIRK) 채널이 설명되어 있습니다. 검정은 GIRK 채널 활동을 측정하는 막 잠재력에 민감한 형광 염료를 활용합니다. 이 기술은 세포 라인의 숫자에 사용하기위한 적응이다.

비디오: A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

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

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

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

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

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

연구

생물학

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 ...

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

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

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

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 ...

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

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

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

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 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 ...

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

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

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

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

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

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

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

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

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

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

Proteoliposome 기반 유출 분석은 CL의 단일 분자의 속성을 확인하는<sup&gt; -</sup&gt; 채널 및 전송기

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

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

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

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

멀티 플렉스 애플리케이션을위한 형광 단백질 유전자 바코드

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

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

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

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

형광 정리 분석을 사용하여 Zebrafish의 신장 기능의 평가

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

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

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

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

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 ...