这项工作得到了美国公共卫生服务奖NS-071530。

1。单元的制备<ol><li>垂体AtT20细胞生长在杜尔贝科的改良Eagle培养基（DMEM培养基）10％马血清和保持在5％ 二氧化碳培养箱的气氛中，在37°C培养为辅。 </li><li>保持文化的传代细胞，每5至7天，使用标准的胰蛋白酶消化过程。 </li><li>被毛黑色，清澈见底的井，50μL聚-L-赖氨酸的96孔板。允许井干在孵化器30分钟。 </li><li>板30000每孔细胞密度和细胞储存在3-4天的孵化器含有200μL的媒体在96板细胞。 </li><li>使用一个愿望设置，组成4公升1塞的一端连接到真空的罐子和一个200μL枪头在另一端，慢慢地吸从单层细胞培养液中。 </li><li>洗细胞与正常缓冲溶液200μL（132 mM氯化钠，氯化钾，1米5毫米中号2氯化钙，氯化镁2 1毫米，5毫米葡萄糖，5毫米肝素钠，用NaOH的pH值7.4）。 </li><li>加入200μL缓冲区包含敏感染料膜电位（MPD），4（3）DiBAC或HLB值021-152（5μM）解决方案，每口井和40分钟的细胞置于37°C。 </li></ol> 2。测试化合物的制备和细胞治疗<ol><li>准备测试化合物的化合物库的连续稀释（股票= 10毫米在DMSO）（96格式）使用1 Versette（ThermoFisher）自动化液体处理系统。 </li><li>取出96孔板，AtT20细胞，从孵化器，并允许板恢复到室温。吸了旧的缓冲区，并适用于新鲜的缓冲溶液中含有细胞的MPD。 </li><li>使用液体处理系统，适用于不同浓度（10纳米到10μm）的测试化合物（2至20μL）和控制解决方案（0.1％二甲基亚砜）（在缓冲溶液containi吴MPD），含有细胞的96孔板。 </li><li>测试化合物荧光测量前5分钟孵育细胞。 </li></ol> 3。激活GIRK通道，荧光测量和分析<ol><li>插入到百特Synergy2荧光酶标仪96孔板，分别获得在激发和发射波长为520和560 nm的荧光背景信号。 </li><li>应用GPCR的配体（生长抑素= 200 nm或卡巴= 10μM）或控制解决方案（单缓冲）（20μL）井（总体积为220μL）使用Synergy2喷油系统激活GIRK通道。收集超过300秒的采样周期10秒的时间间隔，在激发和发射波长为520和560 nm的数据点，分别。 </li><li>获得使用百特GEN5软件的荧光变化的时间过程和峰值幅度，并作进一步的分析数据导出到Excel。 </l我&gt; <li>计算的Z&#39;的因子确定了检测的可靠性。因素Z&#39;决心用板包含2行正面（GPCR的配体）和阴性（单缓冲）控制。 ，Z&#39;因子定义为：Z“= 1 - （3σ+3σ列印 ）/ |μ 的P - 列印 |μ，μ 的P＆μ列印阳性对照和阴性对照信号的手段，和σ 宝洁 Σ 列印阳性对照的标准偏差和负控制信号，分别为6。在0.5至1.0的范围Z&#39;因素表明，检测质量是优良的6。板与0.5 Z&#39;以下因素排除在分析之外。 </li><li>复检中存在BA 2 +或tertiapin - Q调制荧光信号的化合物，以确认内向整流K +通道的特异性。 </li></ol> 4。 ŕ之代表结果使用GIRK通道法测量荧光信号的例子如图2所示。此外GPCR的配体生长抑素（200纳米）的AtT20细胞造成的HLB 021-152荧光信号的一种快速，时间依赖性下降（ 图2）。相比之下，生产控制解决方案，除了在小荧光的瞬时上升，随着时间的推移返回到基线（ 图2）。控制和生长抑素解决方案注入的96孔板峰的荧光值比较了1 Z&#39;因素范围为0.5至0.7。为进一步量化，控制记录减去生长抑素纪录和生长抑素敏感信号分析（ 图2）。 该法提供了一种快速识别药物调节GIRK通道的方法。例如，药物抑制GIRK茶nnel应防止细胞的K +外流减少荧光GPCR的配体介导的跌幅。所示，在图3，治疗的细胞毒素tertiapin-Q块GIRK通道7，产生抑制生长抑素介导的荧光变化。普罗帕酮，抗心律失常药物，在心脏8块GIRK通道，也抑制了荧光的变化（ 图4）。该法也可能是有用确定激活GIRK通道。然而，乙醇的应用（100毫米和200毫米），激活GIRK通道2,3，造成时相比没有显着的变化的荧光信号控制解决方案（P&gt; 0.5）。 <img src="/files/ftp_upload/3850/3850fig1.jpg" alt="图1"/> 图1。GIRK通道荧光检测的实验设计。 AtT20细胞培养在含有膜上的缓冲区Ë电位敏感荧光染料（四）。染料分子进入细胞后，细胞内蛋白质（顶部面板）结合荧光。结合生长抑素（SOM）的GPCR的刺激了G抑制蛋白（七）造成激活GIRK通道（底部面板）。随后外流的K +的细胞，导致膜电位变得更消极的染料分子退出细胞。因此荧光信号的跌幅（底部面板）。 <img src="/files/ftp_upload/3850/3850fig2.jpg" alt="图2"/> 图2。代表HLB值021-152荧光信号AtT20细胞的时间来衡量，此外生长抑素或控制的解决方案。荧光强度的比值（F / F O）的计算除以信号中存在生长抑素（六）（F O）广告前测得的基线信号（或控制解决方案）的生长抑素dition（或控制的解决方案）。每个点代表平均值±SE 5-6井获得。生长抑素或控制解决方案，增加了在零时间（↓）。控制解决方案，除了造成在小瞬态荧光信号的增加。这导致从溶液注射引起的温度变化。 <img src="/files/ftp_upload/3850/3850fig3.jpg" alt="图3"/> 图3。AtT20与tertiapin问（500纳米）的细胞治疗抑制生长抑素结合并阻断GIRK通道的荧光信号介导的跌幅。每个点代表平均值±SE 4-6井获得。生长抑素在零时间（↓）。 <img src="/files/ftp_upload/3850/3850fig4.jpg" alt="图4"/> 图4。与普罗帕酮（20μM）AtT20细胞的治疗，也能抑制在介导生长抑素减少荧光信号。每个点代表平均值±SE 5-6井获得。生长抑素在零时间（↓）。

<ol>
	<li>Hibino, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inward+rectifying+potassium+channels:+their+structure,+function+and+physiological+roles.">Inward rectifying potassium channels: their structure, function and physiological roles.</a> Physiol. Rev. 90, 291-366 (2010).</li><li>Lusscher, C., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+for+G+protein-gated+inwardly+rectifying+potassium+(GIRK)+channels+in+health+and+disease.">Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease.</a> Nat. Rev. Neurosci. 11, 301-315 (2010).</li><li>Kobayashi, T., Ikeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-activated+inwardly+rectifying+potassium+channels+as+potential+therapeutic+targets.">G protein-activated inwardly rectifying potassium channels as potential therapeutic targets.</a> Cur. Pharm. Des. 12, 4513-4523 (2006).</li><li>Terstappen, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+native+and+recombinant+ion+channels+using+a+high-capacity+nonradioactive+rubidium+efflux+assay.">Functional analysis of native and recombinant ion channels using a high-capacity nonradioactive rubidium efflux assay.</a> Anal. Biochem. 272, 149-155 (1999).</li><li>Cheng, C. 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没有利益冲突的声明。

虽然膜电位敏感荧光染料已被用来确定调节离子通道的药物，9,10，这是他们的神经GIRK通道药物发现中的应用的第一次报告。这里GIRK通道荧光检测提供了快速，可靠，实时的方法，筛选配体门控K +通道。与细胞的永生细胞株（HEK293细胞，CHO等）表达一个外生重组GIRK通道11或克隆细胞株（AtT20，PC-12）表达的内源性通道的广泛使用，可以修改该法。此外，该法可适应在胚胎干...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM培养液</td><td> CellGro </td><td> 10-013 </td></tr><tr><td>马血清</td><td> Invitrogen公司</td><td> 16050-114 </td></tr><tr><td> 96孔板</td><td>康宁</td><td> 3603 </td></tr><tr><td>聚-L-赖氨酸</td><td> Sigma-Aldrich公司</td><td> P4707 </td></tr><tr><td>生长抑素</td><td> Sigma-Aldrich公司</td><td> S9129 </td></tr><tr><td>卡巴</td><td> Sigma-Aldrich公司</td><td> C4382 </td></tr><tr><td> HLB值021-152 </td><td> AnaSpec </td><td> 89300 </td></tr><tr><td> versette自动化液体处理程序</td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> synergy2荧光酶标仪</td><td>百特</td><td></td></tr><tr><td> GEN5分析oftware </td><td>百特</td><td></td></tr></tbody></table> 表1。表特定的试剂和设备。

a fluorescent screening assay for identifying modulators of girk channels

G蛋白门控内向整流钾 （GIRK）通道功能广泛的激素和神经递质的细胞介质，并在大脑，心脏，骨骼肌和内分泌组织1,2表示。 GIRK通道成为激活后的配体结合（神经递质，激素，药物等），其细胞膜结合，G蛋白偶联受体（GPCRs）。这种结合使G蛋白（G I GØ），随后结合并激活GIRK通道的刺激。一旦打开了GIRK通道允许的K运动导致细胞静息膜电位变得更负+。因此，GIRK通道激活的神经元减少自发性动作电位的形成和抑制兴奋性神经递质的释放。在心脏，激活GIRK通道的抑制起搏器的活性，从而减缓心率。 <p为开发新的治疗药物，治疗神经痛，吸毒，心律失常和其他疾病3类=“jove_content”的GIRK通道代表的新的目标。然而，这些通道的药理学仍然人迹罕至。虽然一些药物，包括抗心律失常药物，抗精神病药和抗抑郁药阻止GIRK通道，这种抑制是没有选择性的，发生在相对 ​​较高的药物浓度3。 在这里，我们描述了一个实时的筛选试验，确定新的调制GIRK通道。在这个实验中，神经AtT20细胞，表达GIRK通道，装有膜电位敏感荧光染料，如双（1,3 - dibutylbarbituric酸）的三甲oxonol [DiBAC 4（3）]或021-152 HLB值（ 图1 ）。染料分子成为强烈荧光以下的摄取进入细胞内（ 图1）。治疗GPCR的配体细胞刺激GIRK通道打开。由此产生的K +外流的细胞，导致膜电位变得更负，荧光信号降低（ 图1）。因此，GIRK通道通过调节钾离子流出的药物可以用荧光酶标仪测定。不同于其他离子通道筛选试验，如原子吸收光谱法4或放射性分析5，GIRK通道荧光检测提供了一种快速，实时和廉价的甄别程序。

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

一个确定药物相互作用与G蛋白门控内向整流钾实时甄别<sup&gt; -</sup&gt;（GIRK）通道描述。该法采用膜电位敏感荧光染料来衡量GIRK通道活性。这项技术是适应的细胞株上使用。

一个荧光识别GIRK通道调制器的筛选试验

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 次观看.  University of South Carolina, School of Medicine. 一个确定药物相互作用与G蛋白门控内向整流钾实时甄别 -（GIRK）通道描述。该法采用膜电位敏感荧光染料来衡量GIRK通道活性。这项技术是适应的细胞株上使用。

视频: 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.

异源表达在哺乳动物细胞离子通道的突变和功能分析

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

科研

生物学

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

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

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

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

高通量筛选和生物传感与荧光 C。线虫菌株

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

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

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

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

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

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

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

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

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

内向整流钾通道的小分子调节剂的高通量筛选

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.

使用自制双辉光萤光素酶检测的高通量筛选功能

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

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

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

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

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

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</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.

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

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.

遗传条形码与荧光蛋白的复用的应用程序

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.

斑马鱼肾功能评价采用荧光测定间隙

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.

高通量筛选转录后调控基因的化学调节剂

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