この仕事に貢献し、ほとんどが膜構造·機能研究グループからのもので、過去と現在の両方のメンバーが多くの人々があります。すべての我々の暖かい感謝と感謝の意を。この作品は、アイルランド科学財団（07/IN.1/B1836）、国立衛生研究所（GM75915、P50GM073210とU54GM094599）、およびFP7 COSTのアクションCM0902からの助成金によって部分的にサポートされていました。

<ol>
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T., Klukas, O., Saenger, W., Krauss, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+cyanobacterial+photosystem+I+at+2.5+angstrom+resolution.">Three-dimensional structure of cyanobacterial photosystem I at 2.5 angstrom resolution.</a> Nature. 411, 909-917 (2001).</li><li>Grawert, T., Span, I., Eisenreich, W., Rohdich, F., Eppinger, J., Bacher, A., Groll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+reaction+mechanism+of+IspH+protein+by+x-ray+structure+analysis.">Probing the reaction mechanism of IspH protein by x-ray structure analysis.</a> Proc. Natl. Acad. Sci. USA. 107, 1077-1081 (2010).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picolitre-scale+crystallization+of+membrane+proteins.">Picolitre-scale crystallization of membrane proteins.</a> J. Appl. Crystallogr. 39, 604-606 (2006).</li><li>Li, D., Boland, C., Aragao, D., Walsh, K., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+cryo-cooling+crystals+of+membrane+proteins+grown+in+lipidic+mesophases+for+structure+determination+by+macromolecular+crystallography.">Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography.</a> J. Vis. Exp. (67), e4001 (2012).</li></ol>

特別な利害関係は宣言されません。

このビデオの記事では、我々は自動的にタンパク質を含んだ脂質メソフェーズを用いて、96ウェルガラスサンドイッチプレートにメソ結晶化試験でセットアップするロボットを使用する方法を実証した。本研究で使用したロボットは特別にもともと7に記載されているように 、粘性及び粘着性メソフェーズのナノリットルボリュームの正確で再現性の送達のための肯定的な変位のガラスシリンジを含むように設計されていました。 正確さと精度は、ロボットの重要な機能です。しかし、これらの特徴は、ロボットの性能が評価され、キャリブレーションが実行される程度と規則性と同じくらい良いです。それは個々のプレートが設定されている間、ロボットの性能が評価されるべきことは言うまでもない。それはロボットが完璧に機能し、自動で実行するロボットを残していると仮定することは適切ではない。気配りと観察オペレータがposにあるべきitionは音か何かが正常に動作していないと、すぐにそれを修正するために外観によって注意を促す必要があります。 プレートがシールされているとすぐに、それが結晶化試験のために離れて置かれる前に加えて、 各プレートを慎重によく内容の均一性のために目の検査を受けなければならない 。これは、ほんの数秒で実行するようにする必要がありますし、次のプレートをロードしている間に行うことができます。気付かは、例えば、特定のウェルが正しく入力されていないことは確か沈殿分配チップが誤動作しているという事実を強調することができる。メソフェーズの配信は不定期であると見なければならない、関連する障害のある項目は直ちに修正される必要があるでしょう。これらの問題を指摘し、セットの間の関連の調整を行うことにより、貴重な脂質や膜タンパク質など、時間と材料の節約になります。イメージャは、結晶成長、正確さと精度を追跡するために使用されている場合は、撮像中に監視することができます。ドロップまたはボーラスを持つ例えば、体系的な問題イメージャによって場所は、何かが右ではなく、プロトコルのどこか上流の是正措置が必要であることを示す。 信頼性の高い性能故にロボットの利益のために、定期的なスケジュールで校正され、必要に応じなければなりません。校正は、沈殿剤およびウェル内ボーラスと沈殿配置と同様に配信メソフェーズのボリュームを含める必要があります。明らかに、可能な限りキャリブレーションは品質管理運動の対象期間内に実施された試験で使用されるものと同様のボリュームと材料を使用して行われる必要があります。 キャリブレーションと同様に重要なのは、あまりにも在庫がロボットの部品や消耗品の適切な供給を持っているそうです。破局や予期しない障害、閉塞とクラッシュが発生するとを行うことができます。このような場合には、使用可能な交換用沈殿配信先端を有する、例えば、非常に貴重な膜タンパク質調製物は、それが必要ととして使用されていることを意味するかもしれません無駄にされていません。 ロボットの利点のいくつかは、それが本質的に連続的に動作すること、それは苦しんでいないか、または疲労 &quot;が訴える&quot;という事実を含む。ロボットはまた、暗闇の中でのように、制御された照明や環境条件の下で、温度の極端で、人に優しいとは見なされません条件の下で使用することができます。この記事で紹介ロボットはすべての〜20℃周囲条件下で使用されましたしかし、タンパク質および非周囲温度、制御された光11,12及び酸化剤または還元環境13を必要とするプロジェクトがあります。結晶化ロボットが使用されている場合、これらのすべては、比較的容易に、用の仕出し料理することができます。 以前Joveのビデオ記事ではメソメソッド内で結晶化試験は手動で1に設定されている方法を紹介しました。手で確実に分配することができる中間相の最小体積はb限られているyの試験を設定する人の視力と着実利き手。我々の経験では、100 nlの限り低くメソフェーズのボリュームが容易に処理されます。我々は、デフォルトのボリュームが40 NL約手動ディスペンス1研究室を知っている。しかし、かなり小さいボリュームがロボットを使って行うことができます。これとは別に、我々は550ピ ​​コリットルという低メソフェーズボリュームがロボット14分配することができることが示されている。その作品からは、ロボットによる更なる小型化が実施されれば、結晶化実験を行うのに必要な貴重な膜タンパク質の量の大幅な削減につながる、これが可能であることは明らかでした。 このビデオの記事では、3種類の市販のロボットは脂質中間相を用いた高スループット膜タンパク質の結晶化を実証するために使用されていました。 JOVE 1712 1に記載したように、これらの最初のものは、手動で試験を設定する私たちの経験に基づいて、MS＆FBのグループで開発されました。これは楽器ですリーメントは我々に最も精通していると、現在の記事の大半は、その使用に専念しています。他の二つのロボットは、記事が書かれた、関連する映像が、完全性の利益のためにここに含まれている時に、MS＆FBのグループのデモにあった。すべての3つのロボットは非常に同じメソフェーズ分配システム、容積ガラスシリンジ5,7となっている本質的な機能を使用します。彼らは、沈殿剤デリバリーに関して最も大きく異なります。ロボット1と2は、8ウェル、同時に結晶化プレート上の単一の列に同時に沈殿剤を分配することができます。これとは対照的に、ロボット3は、単一のアクションですべての96沈殿ソリューションを分配します。ロボット2は使い捨ての沈殿剤調剤のヒントを持つ唯一の楽器です。特定のアプリケーションに依存する別の楽器に関連付けられている長所と短所がありますが、これらはここで詳しく説明されません。すべての3つの作業とは、私の結晶が得られたことを言えば十分メソメソッド内でmbraneタンパク質。 高分子結晶学による構造決定の全体的なプロセスの次のステップは収穫にとクライオクール結晶にこのビデオの記事で説明されるように設定するプレートから、レコード、それらからプロセスX線回折にしています。これらのトピックについては、このシリーズ1,15で個別Joveの記事で説明されています。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コンポーネント </td></tr><tr><td> Brayer（ローラ） </td><td>フィッシャー·サイエンティフィック</td><td> 50820937 </td><td>ツール</td></tr><tr><td>ガスタイトシリンジ</td><td>ハミルトン</td><td> 81030 </td><td>ツール</td></tr><tr><td>カバーガラス</td><td> Marienfeld </td><td> 01029990911 </td><td>使い捨て</td></tr><tr><td>ガラス板</td><td> Marienfeld </td><td> 1527127092 </td><td>使い捨て</td></tr><tr><td>グリフォンLCPのロボット</td><td>アート·ロビンス</td><td> NA </td><td>ツール</td></tr><tr><td> メソロボットで </td><td> Anachem /ギルソン</td><td> NA </td><td>ツール</td></tr><tr><td>実験ノート</td><td>さまざまな</td><td> NA </td><td>ツール</td></tr><tr><td>蚊LCPのロボット</td><td> TTP LABTECH </td><td> NA </td><td>ツール</td></tr><tr><td>あき両面スペーサテープ</td><td>サンダース株式会社（穴パンチ） </td><td>カスタマイズ</td><td>使い捨て</td></tr><tr><td>沈殿ソリューション</td><td>さまざまな</td><td>さまざまな</td><td>試薬</td></tr><tr><td>精製水</td><td>ミリポア</td><td> NA </td><td>試薬</td></tr><tr><td>雨 - X </td><td>シェルカーケア</td><td> 80199200 </td><td>試薬</td></tr><tr><td>シリンジのヒント</td><td>ハミルトン</td><td> 7770-020（ゲージ22） </td><td>ツール</td></tr><tr><td>組織</td><td>さまざまな</td><td>さまざまな</td><td>使い捨て</td></tr><tr><td>水筒</td><td>さまざまな</td><td> NA </td><td>試薬</td></tr></tbody></table>

use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases

膜タンパク質の構造と機能の研究は大いに高分子X線結晶構造解析（MX）を介して提供タイプの使用可能な高解像度の3次元構造を持っていることから恩恵を受ける。 MXの必須成分は、理想的には回折品質結晶の安定供給である。膜タンパク質の結晶化のメソまたは脂質立方相（LCP）メソッドの中で膜タンパク質を結晶化させるためのいくつかの方法の一つです。これは、結晶を成長するための共連続メソフェーズを利用しています。方法としては、後半のいくつかの壮観な成功を収めており、現在、それを使うことに興味を持って多くの研究グループと注目を集めている。メソッドに関連付けられている課題の一つは、ホスティングメソフェーズが厚い歯磨き粉を彷彿とさせる、非常に粘性と粘着性であるということです。このように、結晶化ウェルに少量で再現可能な方法でそれを手動で調剤することはスキル、忍耐と着実に手を必要とします。柱を行うためのプロトコル膜の構造·機能生物学（MS＆FB）グループ1-3に開発されたstの。方法を説明するJoveのビデオの記事が入手できる1,4です。 メソ試験で設定するための手動のアプローチは、このような結晶の最適化および誘導体化などの特殊なアプリケーションとの明確な利点を持っています。しかしそれは低スループット法であることに苦しむん。ここでは、ロボットメソ結晶化試験で行うためのプロトコルを示しています。ロボットは、スピード、正確性、精度、小型化、還元雰囲気中または低温下、または高温下で、そのような暗闇の中でのように敵対的な条件とみなすことができるもので、長期間にわたって継続的に働くことができるという利点があります。 メソロボットで 、適切に使用すれば、大幅で遅いステップの一つである結晶化を促進することにより、膜タンパク質構造の生 ​​産性と機能の研究を向上させることができます全体的な構造決定のパイプライン。 このビデオの記事では、我々は、 メソ crystallogenesis 内に不可欠粘性及び粘着性メソフェーズを免ずることができる3つの市販のロボットの使用例を示します。最初のロボットは、MS＆FBのグループ5,6で開発されました。他の二つは、最近利用可能になっていると、完全を期すためにここに含まれています。 この記事で取り上げたプロトコルの概要を図1に示されている。すべての操作は室温（約20℃）周囲条件下で行った。

Watch this Scientific Journal Video about Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

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

本明細書では、高分子のX線結晶構造解析を用いた構造決定に使用するための脂質中間相における膜タンパク質のハイスループット結晶化ロボットのアプローチを説明しています。メソッドに不可欠な粘性及び粘着性のタンパク質を含んだ中間相を扱うことができる3つのロボットが導入される。

脂質中間相における膜タンパク質のハイスループット結晶化のためのロボットの使用

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

use-robot-for-high-throughput-crystallization-membrane-proteins

Research

JoVE Journal

Biology

17.4K ビュー.  Trinity College Dublin. 本明細書では、高分子のX線結晶構造解析を用いた構造決定に使用するための脂質中間相における膜タンパク質のハイスループット結晶化ロボットのアプローチを説明しています。メソッドに不可欠な粘性及び粘着性のタンパク質を含んだ中間相を扱うことができる3つのロボットが導入される。

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

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.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

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

生物学

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.

電子線結晶学による構造生物学研究用小型膜タンパク質の二次元結晶化トライアルの評価

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

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

X -線結晶構造のためのタンパク質の結晶化

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

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.

脂質中間相における膜タンパク質の結晶化

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

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

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

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

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.

高分子結晶学による構造決定のための脂質メソフェーズで栽培膜タンパク質の収穫とクライオ冷却結晶

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.

アフィニティータグ組換えタンパク質のハイスループット精製

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 and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

内脂肪量の生化学的および高スループット微視的評価<em&gt;線虫（Caenorhabditis elegans）</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

ウニ​​接合体のハイスループットマイクロインジェクション

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...