我们要感谢博士。詹姆斯Bowie和萨利姆Faham全线Bicelle方法和有益的讨论特拉维夫拉巴斯博士提供的技术专长和指导。我们承认实验支持乐杜。 Rachna Ujwal MemX生物科技有限责任公司，但是，不支持这项工作的财务利益。这项工作是支持部分由美国国立卫生研究院（RO1 GM078844）资助。

全线Bicelle基础的结晶，是由四个基本步骤（图2）:我）准备一个全线Bicelle形成脂质:两亲分子的混合物;二）进入全线Bicelle介质的纯化蛋白质的结合;三）结晶试验（手动或自动机械）;及iv）可视化，晶体的提取和冻结。下面详细介绍这些步骤 1。准备的Bicelles Bicelles可以形成:两亲组合在各种脂质的浓度范围。因此，基于以往的成功条件，初步构成建议（ 见表1） 。最成功的混合物DMPC:CHAPSO全线Bicelle制定，可以购买商业作为预混使用现成的制定（见下面试剂表）或所描述的那样在实验室准备。在本练习中我们将准备在2.8:1的摩尔比1毫升35％DMPC:CHAPSO混合物。 <ol><l我&gt;称取0.26克DMPC（先生677.9克/摩尔），0.09克CHAPSO（先生630.9克/摩尔），并添加去离子水至终体积为1.0 ml。 <ul type="disc"><li>全线Bicelle的比例在10％-40％之间，可以改变一个DMPC:CHAPSO摩尔比从2.6-3.0:1不等（表1）。 </li><li> 注意:全线Bicelle浓度越难以溶解脂质，导致在一个较高的的溶液粘度越高。然而，一个集中的全线Bicelle制定有利的蛋白质浓度低时。 </li></ul></li><li>溶解脂质获得一个同质的解决方案，需要相当大的努力，使这一步的大部分时间消耗在全线Bicelle方法。完全混合通过以下步骤，直到DMPC与周期: <ol><li>温暖的混合物〜40℃水浴或培养箱〜1分钟的旋涡。 <ul type="disc"><li> 注:由于开展更多的周期，气候变暖的混合物将导致在凝胶样CONsistency使得很难以涡。 </li></ul></li><li>酷冰几分钟的旋涡混合物。冷却有助于液化的解决方案，使其更容易，以涡。 <ul type="disc"><li> 注:随着越来越多的循环执行，混合物冷却后可能成为阴天。 </li></ul></li><li>重复上面列出（1.2.1和1.2.2），直到完全溶解脂质的步骤。 <ul type="disc"><li> 注意:此过程可能需要几个小时。全线Bicelle形成表明DMPC与相行为的变化:CHAPSO制定。建成后，将混合物在室温或以上的透明凝胶上冰的粘稠液体。 </li></ul></li></ol></li><li>全线Bicelle的混合物，现在可以使用，并保持长期存储（长达5年的），可在-20 ° C。由于水解磷脂头组的风险，这是不可取的，储存在室温下长时间bicelles。 </li></ol> 2。蛋白质团到bicelles 从bicelles获得大多数的MP结构结晶DMPC:CHAPSO全线Bicelle浓度从2％至8％不等的8至12毫克/毫升（ 见表1）的蛋白浓度。如果可能的话，初始屏幕应使用这些准则和额外的浓度可在优化阶段筛选。 LCP的方法相比，蛋白与bicelles法团是一个简单的过程（ 图3），它应该做的结晶试验的同一天。 <ol><li>解冻DMPC:CHAPSO全线Bicelle相位的变化，以明确的凝胶在室温下混合，直到。 <ul type="disc"><li> 注:多个冷冻解冻不会影响全线Bicelle行为。 </li></ul></li><li>放在冰的混合物，液化和简要涡重新建立一个同质全线Bicelle阶段。当置于冰上的混合物可能变得浑浊。 </li><li>神父OM这一点上，保持全线Bicelle混合物和纯净冰蛋白。这将保持在液体中的相适合吹打全线Bicelle。 </li><li>全线Bicelle混合物添加洗涤剂溶解在1:4（V / V）的比例纯化蛋白。 <ul type="disc"><li>例如:100μL蛋白全线Bicelle混合获得混合与20μL全线Bicelle 80μL蛋白。如果蛋白浓度为15毫克/毫升，全线Bicelle浓度为35％，这会给一个全线Bicelle注册的蛋白质混合物的蛋白浓度12毫克/毫升和7％的全线Bicelle浓度。 </li></ul></li><li>轻轻吹打的内容向上和向下，直到溶液变得清晰和均匀混合。 <ul type="disc"><li> 注:如果气泡出现，快速自旋与台式离心机（30-60秒，13000 RPM，4℃），可以帮助将其删除。 </li></ul></li><li>在冰上孵育至少30分钟的混合物，以促进蛋白质的INT的完整纳入Ø bicelles。现在的蛋白质全线Bicelle混合结晶试验的准备。 </li></ol> 3。设置结晶试验其他脂质像LCP的结晶技术需要专门的设备，由于介质的高粘度;，但bicelles独特的相行为允许在执行几乎任何标准的结晶格式，包括机器人（图3）。悬挂或坐滴格式，使用标准的市售屏幕可以进行结晶试验。 <ol><li>无论是设立托盘手动或使用结晶机器人，保持在冰上的蛋白质，全线Bicelle混合物。这将保持全线Bicelle的蛋白质混合物冷，确保溶液的粘度是最小的。 </li><li>手册结晶试验 - 使用标准的吸管，蛋白质全线Bicelle混合物可以以同样的方式与水库解决方案混合作为正常执行教育署可溶性或洗衣粉溶解膜蛋白。 <ul type="disc"><li> 注:在审判全线Bicelle蛋白混合物置于冰上。 </li></ul></li><li> 机器人结晶试验 -我们有专门适合蚊子的结晶机器人这些试验，但原则（以下相同的预防措施）的技术应与所有的结晶机器人兼容。以下提示将确保全线Bicelle蛋白混合保持冷静和准确地由机器人吸管: <ol><li>预冷板，放在冰的蛋白质，全线Bicelle混合物。 </li><li>移液器进入板块全线Bicelle蛋白混合，并继续保持在冰板。这个板块应该开始运行之前的最后一个项目上的机器人。 </li><li>设置水库托盘和结晶盖平台3和5分别蚊子机器人，。 </li><li>将含有蛋白全线Bicelle混合平台4板蚊子的机器人。这可以保证蛋白质，全线Bicelle混合物最后被拾起机器人，并立即释放。 </li><li>为了防止加热和粘度增加，不混合蛋白全线Bicelle混合水库。 </li><li>执行多个屏幕上时，立即返回全线Bicelle蛋白质板冰的冷却尽快完成运行。 </li></ol></li><li>可以选择滴体积比（蛋白质:水库）作为传统的结晶试验。例如，可以成立的初步水晶试验，用蚊子机器人，使用0.25μL蛋白:全线Bicelle混合加0.25μL水库。 </li><li>孵育晶体试验室，在20 ° C。因为bicelles阶段行为是取决于温度，温度是一个很好的筛选和优化参数。 <ul type="disc"><li>较高的温度，诱发层状第12期（ 图1），其中已预先组织的优势层中的蛋白质。温度低于20 ° C可以进行筛选，但你不应该低于4 ° C可能，因为这导致脂质过长时间的沉淀。 </li></ul></li><li>在相同的方式作为传统的结晶试验，全线Bicelle试验应在晶体的外观和增长的定期监测。我们建议检查托盘上的第1和第3天安装后每周检查。 </li><li>水晶可以进行优化，使用洗涤剂，包括网格筛选，添加剂筛选，温度变化等。此外，全线Bicelle百分比和蛋白质晶体的基础经常使用的方法:全线Bicelle的比例可以是多种多样的。此外，bicelles可以掺杂特定血脂，可能是蛋白质稳定性或功能所必需的。 </li></ol> 4。可视化，晶体的提取和冻结由于与蛋白质，全线Bicelle混合物的晶体试验<html>粘度类似蛋白洗涤剂滴，可视化和晶体提取是家常便饭，像传统的设置进行。 <ol><li>可视化:在LCP的媒体，通常需要高品质的照明下正常和极化晶体检测，可视化是不bicelles阻碍。有色以及无色蛋白晶莹的水珠，可以很容易地使用标准的显微镜，无需特殊设备需要进行分析。 </li><li> Bicelles，其他油脂媒体一样，往往会产生高比例的误报。一个UV镜，大大有助于区分盐晶体蛋白（图4）。 </li><li>提取和冷冻:水晶提取和冷冻是相对简单，并且不需要解散周围全线Bicelle媒体。此外，全线Bicelle阶段本身提供了一些温和的冷冻保护。 </li></ol> 5。代表性的成果: <html> NT"一般需要2-3天晶体出现，大约一周或更长时间为他们成长到他们的最大尺寸，这是细菌视紫红质和鼠标的电压依赖性阴离子通道1（mVDAC1） 晶体 4,8对于其他的膜蛋白，它可以需要几个星期，晶体生长，所以重要的是继续监测，远远超出了第一周的晶体试验。 油脂与其他媒体中，bicelles往往以形成形状可能出现结晶。它也被观察到，他们带领到更高比例的盐和洗涤剂晶体。紫外线-色氨酸荧光显微镜检测可以显着帮助消除这类非蛋白质的误报。图4显示了脂质形状，盐和蛋白质晶体在可见光和紫外线光的效果，以帮助区分不同的结果可观察到的。 83fig1.jpg"/&gt; 图1。全线Bicelle示意图 。 DMPC（蓝色）和CHAPSO（绿色），保护双层的疏水边缘，如两亲如形成一个双层脂质分子组成Bicelles。随着温度的增加，圆盘状bicelles经历了一个相变成穿孔层状表12。 <img alt="图2" src="/files/ftp_upload/3383/3383fig2.jpg" /> 图2。全线Bicelle结晶的方法，概述了四个基本步骤的流程图。 <img alt="图3" src="/files/ftp_upload/3383/3383fig3.jpg" /> 图3。晶体试验设置示意图。纯化洗涤剂溶解膜蛋白可以直接混入的内容由简单的移液一起上冰bicelles。冰〜30分钟，结晶试验后孵化的蛋白质/全线Bicelle混合物可以设置使用任何标准格式，包括机器人。 <img alt="图4" src="/files/ftp_upload/3383/3383fig4.jpg" /> 图4。晶体试验的可视化。可见光图像（ 顶部面板 ）和紫外线图像（ 底部面板 ）（ 一 ）针形观察到晶体中盐的唯一条件。可以检测无荧光的晶体，假阳性的迹象。 （二）在MPD条件杆状结晶形成。晶体荧光弱，但被发现使用X射线衍射的非蛋白质。 （三）水晶观察到约四个星期后，设立审判。紫外光下的强荧光，证实它是一种蛋白质晶体。 <table border="1"><tbody><tr><td> 号 </td><td> 蛋白质 </td><td> 来源 </td><td> 全线Bicelle配方 </td><td> 蛋白质Concentrati上</td><td> 洗涤剂1 </td><td> 决议案（a） </td><td> 参考 </td></tr><tr><td> 1 </td><td>细菌视紫红质2 </td><td> Halobacterium salinarum </td><td> 8％DMPC:CHAPSO（2.8:1） </td><td> 8毫克/毫升</td><td></td><td> 2.0 </td><td> Faham和鲍伊，2002年</td></tr><tr><td></td><td></td><td></td><td> 8％DTPC:CHAPSO（3:1） </td><td> 8毫克/毫升</td><td></td><td> 1.8 </td><td> Faham 等，2005年</td></tr><tr><td> 2 </td><td> β2-肾上腺素受体/晶圆厂复杂</td><td> 智人 </td><td> 8.3％DMPC:CHAPSO（3:1） </td><td> 10毫克/毫升</td><td> DDM </td><td> 3.4/3.7 </td><td> Rasmussen 等人，2007年</td></tr><tr><td> 3 </td><td>电压依赖性阴离子通道1 </td><td> 小家鼠 </eM&gt; </td><td> 7％DMPC:CHAPSO（2.8:1） </td><td> 12毫克/毫升。 </td><td> LDAO </td><td> 2.3 </td><td> Ujwal 等，2008 </td></tr><tr><td> 4 </td><td> Xanthorhodopsin </td><td> Salinibacter曲霉 </td><td> 4.2％DMPC，新墨西哥州5％ </td><td> 4毫克/毫升。 </td><td> DDM </td><td> 1.9 </td><td> Luecke 等。 </td></tr><tr><td> 5 </td><td>菱形蛋白酶</td><td> 大肠杆菌 </td><td> 2％DMPC:CHAPSO（2.6:1） </td><td> 9毫克/毫升</td><td>壬基酚葡萄糖苷</td><td> 1.7 </td><td> 2011年Vinothkumar， </td></tr></tbody></table> 1用于膜蛋白的纯化的洗涤剂 2从紫膜的母语血脂可能在纯化过程中同时进行 表1。膜蛋白结构的结晶条件总结解决使用 bicelles 。

<ol>
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没有利益冲突的声明。

Bicelles是一个独特的，提供原生双层式的环境，而表现犹如洗涤剂溶解油脂媒体。此属性，给出了bicelles一个明显的优势超过其他脂质体的结晶方法，因为没有学习曲线，这种技术所需的专用设备。一旦bicelles，无论是商业或准备在实验室中，他们可以直接与纯化的蛋白质，从这个角度混合结晶试验几乎完全与标准洗衣粉的协议进行的。此外，bicelles提供了包括与其他技术相比，延长贮存期，蛋白?...

high-throughput crystallization of membrane proteins using the lipidic bicelle method

在许多生理过程，如抽水整个否则周围所有的细胞和细胞器的防渗膜双层的具体分子膜蛋白（MPS）发挥了关键作用。 MPS导致许多人类的疾病和失调的功能发生变化，因此，其结构复杂的认识，仍然是生物学研究的重要目标。然而，国会议员的决心结构仍然是一个重大的挑战往往来自其疏水性所引起的。 国会议员的双层内嵌入大量的疏水区域。经常使用的洗涤剂这些蛋白质溶解产生一种蛋白质，然后可以在一个类似的方式为可溶性蛋白操纵洗涤剂胶束双层。传统上，进行结晶试验使用一种蛋白质清洁剂的混合物，但他们往往抵制结晶或生产质量较差的晶体。出现这些问题，因洗涤剂无法充分模仿双层导致稳定性差和异质性。此外，洗涤剂盾牌疏水表面的MP减少晶体接触的表面积。来规避这些弊端国会议员可结晶油脂的媒体，更加紧密地模拟其内源性环境，并在最近成为了MP的结晶新技术。 油脂立方米阶段（LCP）是一个立体的脂质双层渗透 1水通道的互联系统。虽然monoolein是选择脂质，如monopalmitolein和monovaccenin相关的血脂也被用来做LCP 的 2 。国会议员被纳入LCP的，他们在三个层面和饲料晶核弥漫。 LCP的一个巨大优势是，该蛋白在更自然的环境中仍然存在，但该方法具有技术的弊端，包括高VISCosity（需要专门的仪器），并在晶体中的可视化和操纵3,4的困难。由于这些技术上的困难，我们利用另一个结晶bicelles 5,6（ 图1）油脂中。 Bicelles是与两亲（CHAPSO）或短链的脂质（DHPC）一个混合的磷脂酰胆碱脂质（DMPC）所形成的脂质/两亲分子的混合物。在每个全线Bicelle光盘，脂质分子生成双层而行两亲分子的非极性边缘的双层和洗涤剂提供有益的特性。重要的是，其转变温度以下，蛋白全线Bicelle混合物粘度降低和控制在一个类似的方式溶解洗涤剂，国会议员，bicelles兼容结晶机器人。 Bicelles已成功地用于结晶几个膜蛋白5,7-11（见表1）。这种增长的集合蛋白质演示的bicelles结晶从原核和真核来源的α-螺旋和β表国会议员的多功能性。由于这些成功和高通量实施简单，bicelles应该是每一个膜蛋白晶体学的武器库中的一部分。在这个视频中，我们描述全线Bicelle方法，并提供一个一步一步的协议，使用标准的机器人纯化国会议员成立的高通量结晶试验。

Watch this Scientific Journal Video about High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method at JoVE.com

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Bicelles脂质/两亲分子的混合物保持在脂质双层膜蛋白（MPS），但有独特的相行为，有利于高通量筛选的结晶机器人。这项技术已成功地生产从原核和真核来源的高分辨率结构。本视频介绍了生成油脂全线Bicelle混合物，纳入全线Bicelle混合物的国会议员，成立从中长期的结晶试验（手动以及作为机器人）和收割晶体的协议。

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

high-throughput-crystallization-membrane-proteins-using-lipidic

Research

JoVE Journal

Biology

23.6K 次观看.  University of California Los Angeles. Bicelles脂质/两亲分子的混合物保持在脂质双层膜蛋白（MPS），但有独特的相行为，有利于高通量筛选的结晶机器人。这项技术已成功地生产从原核和真核来源的高分辨率结构。本视频介绍了生成油脂全线Bicelle混合物，纳入全线Bicelle混合物的国会议员，成立从中长期的结晶试验（手动以及作为机器人）和收割晶体的协议。

视频: High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...