我们感谢美国国立卫生研究院（1R21CA138373站起来，对这项工作的财政支持（SU2C）癌症。HY是感激，2009年亿利奖由美国癌症研究，从悉尼金梅尔基金会金梅尔学者奖协会癌症研究中心（SKF - 08 - 101），和美国国家科学基金会教师早期职业奖（NSF0954819）。

1。克隆的注意事项<ol><li> NheI和BamHI酶切位点和5&#39; -磷酸两侧的利益的战区导弹防御系统的商业化准备寡核苷酸可以连接到pTox7（我们实验室的一个碱基对的插入，修改后直接酶 BamHI酶切位点14）（图3）消化顺序用BamHI和NheI。寡核苷酸是一个例子所示： 5&#39;ctagcTMDSEQUENCEg3“ 3“gTMDSEQUENCEcctag5” </li></ol> TMD的顺序应该是12-24残基（短序列大概拉长矢量编码的疏水残基）。为了调查的接口，顺序残留的插入和随之而来的残留删除ToxR的战区导弹防御系统的相对旋转的结果 。15,16最后，阿拉伯糖的浓度应在0.001和0.01％之间不同，应建立战区导弹防御系统的设计的4个变种（W / V）来确定观察到的最大的差异是在不同的战区导弹防御系统的序列之间的β-半乳糖苷酶信号的浓度;测试不同表达水平的建议，以确定下可以区分不同的亲和力最好的条件。除了阿拉伯糖和抗生素，IPTG的0.4毫米，可用于增强亲和力的差异不同的TMDS。 ToxR测量应至少一式四份。整个过程应重复至少三次不同质粒转换。 2。生长的细菌培养<ol><li>轻轻解冻FHK12主管冰和转移到15 ml培养管细胞（200μL）。加入质粒DNA（200纳克）和细胞在冰上孵育30分钟。 </li><li>热休克细胞孵育90小号在42℃，孵育2分钟冰。 </li><li>添加SoC媒体（800μL），并在37样品°摇晃一小时（300转）彗星</li><li>氯霉素（30微克/毫升）和阿拉伯糖（0.0025％W / V）一式三份在15毫升培养管改造的混合物50μL接种5毫升LB培养基。孵育样品在37℃，摇晃20小时（300转） （或者5μL文化可以被用来在96孔板接种100μL介质处理大量样品时，这种方法非常有用，但错误会稍高。为了避免蒸发，这将导致错误，填补与媒体的最外层的水井，但不使用他们的样品，最后，双包装盖子和封口膜板之间的联合）。 </li></ol> 3。测量的β-半乳糖苷酶的活性<ol><li>板阅读器预热至28 ° C。 </li><li>转移到一个水库的Z -缓冲，具有较大的枪头，确保只占用上层（水层）。将一个96孔板的孔100μL新鲜准备Z-buffer/chloroform。转移到板一式四份井5μL每一种文化。忽略文化的四口井将作为空白。 </li><li>测量外径595 ​​的板，以确定细胞密度。 </li><li>加入50μL的Z-buffer/SDS板的所有油井。 10分析细胞板，摇板阅读器。确保细胞悬浮液是明确的裂解后，如果需要重复晃动一步。不完全溶解建议Z-buffer/chloroform是不新鲜配制。 </li><li>加入50μL新鲜配制Z-buffer/ONPG所有的井，并返回该板块的酶标仪和测量外径405每20分钟30秒。 </li><li>在使用下列公式（记住要减去空白）计算β-半乳糖苷酶的活性。外径405 /分的比例应该是使用所有数据点的OD 405范围0.0到1.0采用线性模型的拟合计算。 <img src="files/ftp_upload/2721/2721eq1.jpg" alt="公式1" /> 米勒的单位不同，有时在不同的日子记录。因此，这样的全球行动纲领“的参考构造应在每个测试测量。其值可用于ToxR值正常化。 </li></ol> 4。蛋白表达的控制<ol><li>执行免疫印迹验证甚至之间的结构蛋白的表达。结合在离心50μL一式三份的文化和离心（2000转，4分钟）。吹打取出上清，重悬在2个样品上样缓冲液的残留颗粒。 </li><li>在一个标准的8％凝胶负载7.5μL进行电泳125 V为1小时5分钟。传输后，孵化，与抗- MBP HRP标记抗体和可视化;嵌合蛋白有时看到一些大约48 kDa的降解产物在约70 kDa的观察。内源性的MBP也观察到在45 kDa的（见图5）。 </li></ol>5。控制适当的膜插入麦芽糖结合蛋白缺失的细胞系在使用，以评估适当的膜插入嵌合的战区导弹防御系统的构造。当最小的媒体作为唯一碳源生长与麦芽糖，只有正确位于周质麦芽糖结合蛋白的表达不可或缺的一种膜表达产物能够成长。 <ol><li>变换PD28细胞（如FHK12细胞所述），接种2毫升LB培养基。细胞增长37 ° C的晃动（300转）过夜。 </li><li>在3500转离心沉淀细胞，10分钟，4 ° C和再悬浮在PBS（2毫升）洗轻柔吹打一个大型尖或温和的震荡。颗粒的细胞（如上述），第二次用PBS洗，颗粒，最后悬浮在PBS（1毫升）。 </li><li>使用25μL重悬细胞接种5毫升，一式三份，并培育基本培养基，在37 °（300转）用颤抖的彗星。 15-25小时，大约每2小时转移到96孔板和使用的板块，读者的阅读每个样品200μLOD 595读数。 </li></ol> 6。代表性的成果： ToxR转录报告基因检测分析如图4所示的跨膜结构域的寡聚化倾向使用的一个例子。以前，我们进行了调查跨膜齐聚的multispanning膜的组成蛋白质的潜伏膜蛋白1（LMP - 1）通过各种技术，包括ToxR 14个跨膜结构域五（TM5）表现出强烈的倾向oligomerize的;这是证明高米勒单位，与阳性对照，GPA，以及建立dimerizing序列。一个有害突变，D150A，TM5降低oligomerize序列的能力。 LMP - 1的TM1不显着oligomerize和展品一个非常低的米勒单位略高于空白，非FHK12细胞转化的信号，信号。 <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="图1" /> 图1。卡通描绘ToxR记者分析。 ToxR和LacZ基因转录的激活二聚体跨膜结构域（TMD）的驱动齐聚结果。 LacZ基因产物，β-半乳糖苷酶可以量化作为衡量oligomerize一个战区导弹防御系统的倾向。 <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="图2" /> 图2。生产的光吸收物种O - nitrophenolate（ONP）的β-半乳糖苷酶结果的ONPG水解断裂。 <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="图3" /> 图3。pToxR7质粒图谱。 <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="图4" /> 图4。代表ToxR转录报告基因检测分析潜伏膜蛋白1的跨膜结构域的寡聚化倾向。跨膜结构域5（TM5）oligomerizes强烈，而跨膜结构域1（TM1）展品只有弱相互作用。 TM5 D150A突变显着降低能力oligomerize。 GPA是作为一个强大的二聚体阳性对照序列。空白表示未转换FHK12细胞。 <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="图5" /> 图5。蛋白表达的免疫印迹。 <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="图6" /> 图6。PD28互补的检测，以控制正确的膜插入到周质。阴性对照代表缺乏麦芽糖结合蛋白的构造。

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ToxR转录报告基因检测是一个浅显的方式，以确定有oligomerize潜力的跨膜序列。由于内的细菌内膜发生的相互作用，这个实验绕过膜模仿的环境中学习系统的有效性相关的问题。这个实验，克隆到一个单一的质粒多个TMDS可以很容易地并行执行，并可以进行整个实验在96孔板格式，可用于高通量分析蛋白质序列的大量。一旦17已检测到的相互作用，可关键的功能残留审问突变分析，使所涉及?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> BamHI位的限制酶</td><td> Invitrogen公司</td><td> 15201023 </td><td> Invitrogen公司的酶被发现比其他供应商更有效率</td></tr><tr><td> NheI限制酶</td><td> Invitrogen公司</td><td> 15444011 </td><td> Invitrogen公司的酶被发现比其他供应商更有效率</td></tr><tr><td> 15 mL培养管</td><td> Fisher Scientific则</td><td> 14 - 956 - 1J </td><td></td></tr><tr><td> SoC媒体</td><td> Teknova </td><td> S0225 </td><td>合适的音量和高压灭菌消毒。 </td></tr><tr><td> LB培养基</td><td> Sigma - Aldrich公司</td><td> L7275 </td><td>合适的音量和高压灭菌消毒。 </td></tr><tr><td>氯霉素</td><td> Sigma - Aldrich公司</td><td> CO 3的 78 </td><td>存放在冰箱的库存为30毫克/毫升的溶液中乙醇</td></tr><tr><td>阿拉伯糖</td><td> Fluka公司</td><td> 10839 </td><td>存放在冷冻水（W / V）的2.5％股份的解决方案</td></tr><tr><td> NA 2 HPO 4 </td><td> Sigma - Aldrich公司</td><td> S9390 </td><td></td></tr><tr><td>的NaH 2 PO 4 </td><td> Sigma - Aldrich公司</td><td> S9638 </td><td></td></tr><tr><td>氯化钾</td><td> Mallinckrodt化学品</td><td> 6858-06 </td><td></td></tr><tr><td>硫酸镁4 .7 H 2 O </td><td> Sigma - Aldrich公司</td><td> 63138 </td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> Sigma - Aldrich公司</td><td> L6026 </td><td></td></tr><tr><td> 2 - 硝基苯β- D -半乳糖苷（ONPG） </td><td> Sigma - Aldrich公司</td><td> 73660 </td><td></td></tr><tr><td> Z -缓冲</td><td></td><td></td><td> 16.1克钠2 HPO 4 5.5克的NaH 2 PO 4 0.75克氯化钾 0.246克硫酸镁4 化妆至1 L，pH值7.0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200毫升的β-巯基乙醇，2 mL氯仿，补至20 mL，与Z -缓冲。涡1分钟，1分钟800转的离心机。做出新的每块板。 </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160毫克SDS溶解于10 mL Z -缓冲</td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40毫克ONPG于10 mL Z -缓冲。做出新的每块板</td></tr><tr><td> β-巯基乙醇</td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td>抗- MBP单克隆抗体（HRP标记） </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td>最小媒体与麦芽糖</td><td></td><td></td><td> 1个M9的盐，0.4％麦芽糖，1毫克/毫升硫胺素，2毫米硫酸镁 </td></tr><tr><td> 96孔平底板</td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td>板阅读器</td><td> Beckman Coulter公司</td><td> DTX880模检测器</td><td></td></tr><tr><td>水浴</td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td>摇床</td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

蛋白质跨膜结构域的过于简单化的看法只是在磷脂双层锚早已disproven。跨膜蛋白在许多情况下，已经发展了高度复杂的行动机制。1-3的一种方式在膜蛋白可以调节自己的结构和功能是疏水螺旋的直接和具体的联系，形成结构化的跨膜低聚物4,5近来工作的重点分布在膜环境优先氨基酸在水溶液和不同的分子间作用力，驱动蛋白协会。6,7然而，在跨膜结构域的蛋白质的分子识别研究仍落后于那些滞后相比水溶性地区。一个主要的障碍依然存在：尽管跨膜齐聚可以实现显着的特异性和亲和力，他们协会的8直接测量是具有挑战性的。可应用的整合膜蛋白的功能研究的传统方法阻碍了固有的不溶性考试序列。生物物理研究代表跨膜结构域的合成肽获得的见解可以提供有用的结构的洞察力。然而，洗涤剂胶束或在这些研究中用来模仿细胞膜的脂质体系统的生物相关性往往是质疑;做肽在这些条件下采取类似母语的结构和其功能的行为，真正体现内的原生膜的行动模式？为了研究天然磷脂双层的跨膜序列之间的相互作用，Langosch实验室开发ToxR转录记者分析。利益的跨膜结构域嵌合蛋白与麦芽糖结合蛋白的位置，周质和ToxR表示提供的一份报告齐聚水平（图1）。 在过去的十年中，几个其他团体（如Engelman，DeGrado，夏嘉曦）进一步优化和应用这ToxR记者分析。10-13各种ToxR检测已成为金标准，以测试在细胞膜蛋白质相互作用。我们在此展示一个典型的实验操作，在我们的实验室，主要如下Langosch开发协议中进行。这个普遍适用的方法是非常有用的跨膜结构域自我协会在大肠杆菌中的分析大肠杆菌 ，其中β-半乳糖苷酶的生产是用来评估TMD齐聚倾向。战区导弹防御系统，诱导二聚化后，ToxR结合造成的β-半乳糖苷酶的LacZ基因调节的 CTX发起人。 ONPG除了lyzed细胞是通过比色读数。生产的光吸收物种O - nitrophenolate（ONP）（图2）中的β-半乳糖苷酶结果的ONPG水解断裂。

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

一个高效的程序，以评估单通跨膜结构域的寡聚化倾向（TMDS）描述。融合到ToxR的战区导弹防御系统组成的嵌合蛋白在大肠杆菌记者应变中表达。 TMD诱导齐聚的原因ToxR，记者​​蛋白的转录和生产，半乳糖苷酶的激活二聚体。

跨膜结构域齐聚倾向ToxR含量决定

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

transmembrane-domain-oligomerization-propensity-determined-toxr

Research

JoVE Journal

Biology

15.1K 次观看.  University of Colorado at Boulder. 一个高效的程序，以评估单通跨膜结构域的寡聚化倾向（TMDS）描述。融合到ToxR的战区导弹防御系统组成的嵌合蛋白在大肠杆菌记者应变中表达。 TMD诱导齐聚的原因ToxR，记者​​蛋白的转录和生产，半乳糖苷酶的激活二聚体。

视频: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

科研

生物学

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估：兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

研究内质网和线粒体相互作用通过<em&gt;原位</em&gt;接近连接分析在固定细胞

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...