هناك العديد من الذين ساهموا في هذا العمل ومعظمها من الغشاء الهيكلية والوظيفية المجموعة الأحياء، وكلاهما عضو في الماضي والحاضر. لجميع نتقدم بأحر الشكر والتقدير. وأيد هذا العمل في جزء من المنح المقدمة من مؤسسة العلوم ايرلندا (07/IN.1/B1836)، والمعاهد الوطنية للصحة (GM75915، وP50GM073210 U54GM094599)، وFP7 CM0902 العمل COST.

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الإعلان عن أي تضارب في المصالح.

في هذه المقالة الفيديو وأثبتنا كيفية استخدام الروبوت لتعيين تلقائيا في التجارب تبلور في المتوسط ​​96-وحات جيدة ساندويتش الزجاج باستخدام mesophase البروتين شحمي لادن. وقد صممت خصيصا للروبوتات المستخدمة في هذا العمل ليشمل الزجاج الإزاحة الموجبة للتسليم حقنة دقيقة وقابلة للتكرار وحدات التخزين نانولتر من mesophase لزج ودبق، كما هو موضح في الأصل 7. الدقة والدقة هي السمات الهامة لالروبوت. ومع ذلك، وهذه الخصائص هي فقط جيدة بقدر درجة وانتظام التي يتم تقييم أداء الروبوت ويتم تنفيذ المعايرة. فمن نافلة القول أنه ينبغي تقييم أداء الروبوت في حين أنه يتم حاليا وضع لوحات فردية. من غير المناسب أن نفترض أن الروبوت سوف تعمل لا تشوبه شائبة وترك الروبوت لتشغيل غير المراقب. يقوم مشغل اليقظة وملاحظ تكون في نقاط البيعition أن نلاحظ من خلال الصوت أو ظهور عندما يكون هناك شيء لا يعمل بشكل صحيح وتصحيح على الفور. بالإضافة إلى ذلك، ينبغي تفقد كل لوحة بعناية من قبل العين توحيد محتويات جيدا بمجرد اغلاق لوحة وقبل وضعه بعيدا عن المحاكمات التبلور. وينبغي أن يتطلب سوى بضع ثوان لأداء ويمكن القيام به في حين يتم تحميل لوحة القادم. يلاحظ، على سبيل المثال، قد لا يتم بشكل صحيح ملء الآبار خاصة تسليط الضوء على حقيقة أن بعض تلميح الاستغناء الكثيرمن تعمل بصورة صحيحة. يجب تسليم mesophase أن ينظر إليها على أنها غير منتظمة، فإن البند ذي الصلة خلل تحتاج إلى تصحيح على الفور. بالإشارة إلى هذه القضايا وإجراء التعديلات ذات الصلة خلال مجموعة سيوفر وقتا والمواد، بما في ذلك البروتين والدهون قيمة الغشاء. إذا تم استخدام تصوير لتتبع نمو البلورات، ويمكن أيضا أن دقة ودقة رصد خلال التصوير. على سبيل المثال، مشاكل منهجية مع انخفاض أو البلعةالموقع من قبل تصوير تشير إلى أن شيئا ما ليس صحيحا وأنه يلزم اتخاذ إجراءات تصحيحية المنبع في مكان ما في البروتوكول. في مصلحة الأداء الموثوق ذلك يجب معايرة الروبوت على جدول منتظم وحسب الحاجة. يجب أن تشمل المعايرة حجم مرسب وmesophase تسليمها وكذلك التنسيب بلعة ومرسب في البئر. من الواضح، ينبغي أن يتم معايرة حيثما كان ذلك ممكنا باستخدام وحدات التخزين والمواد مماثلة لتلك التي سيتم استخدامها في التجارب التي أجريت في الفترة التي يغطيها نوعية ممارسة السيطرة. بنفس أهمية المعايرة وذلك أيضا هو وجود في سوق الأسهم إمدادات كافية من قطع الروبوت واللوازم. يمكن فشل كارثي وغير متوقعة، وانسداد حوادث والقيام تحدث. في مثل هذا الحدث، وكان يتوفر على معلومات سرية استبدال تسليم الكثيرمن، على سبيل المثال، قد يعني أن يتم استخدام البروتين الغشاء قيمة للغاية كما يجب إعداد ولا يضيع. بعض من مزايا الروبوت تشمل حقيقة أنه سوف يعمل بشكل مستمر في الأساس، وأنه لا يعاني من أو &quot;يشكون من&quot; التعب. ويمكن أيضا أن تستخدم الروبوت في ظل ظروف التي لا تعتبر الإنسان والعائلات، كما هو الحال في الظلام، تحت اضاءة مسيطر عليها والظروف البيئية، وعلى اقصى درجة الحرارة. واستخدمت جميع الروبوتات موضح في هذه المقالة في ظل الظروف المحيطة في C. ~ ° 20 ومع ذلك، هناك بروتينات والمشاريع التي تتطلب درجات حرارة غير المحيطة، وعلى ضوء سيطرة 11،12 والمؤكسدة وتقليل أو بيئة 13. يمكن كل هذه لبيت، بسهولة نسبية، عند استخدام الروبوت التبلور. في مقال سابق إن الرب فيديو أثبتنا كيف يتم تعيين التجارب التي تبلور في طريقة المتوسط ​​يصل يدويا 1. ويقتصر حجم الحد الأدنى من mesophase التي يمكن الاستغناء موثوق باليد بذ البصرية وحدة ثابتة الإنصاف للشخص إقامة المحاكمات. في تجربتنا، يتم التعامل مع بسهولة حجم mesophase منخفضة تصل إلى 100 NL. ونحن نعرف من أحد المختبرات حيث حجم الافتراضي يدويا الاستغناء حوالي 40 NL. ومع ذلك، حجم أصغر بكثير ممكنة باستخدام الروبوت. بشكل منفصل، وقد أظهرنا أنه يمكن الاستغناء عن كميات منخفضة mesophase إلى 550 picoliters آليا 14. من أن العمل كان من الواضح أن المزيد من التصغير الروبوت المحتملة التي، إذا ما نفذت، من شأنه أن يؤدي إلى انخفاض كبير في كمية البروتين الغشاء قيمة اللازمة لإجراء المحاكمة التبلور. في هذه المقالة الفيديو، تم استخدام ثلاثة الروبوتات المتاحة تجاريا لإثبات عالية الإنتاجية بلورة البروتين غشاء شحمي باستخدام mesophases. تم تطوير أول هذه المجموعة في الفيس بوك وMS بناء على خبرتنا إقامة محاكمات يدويا، كما هو موضح في 1712 إن الرب 1. هذا هو الصكوكمنة لدينا أكثر ألفة مع ويخصص الجزء الأكبر من المادة الحالية لاستخدامها. كانت الآخران الروبوتات على مظاهرة في مجموعة الفيس بوك وMS في وقت كتابة المقال ويتم تضمين لقطات ذات الصلة هنا في مصلحة اكتمالها. جميع الروبوتات تستخدم ثلاثة منها كثيرا نفس الاستغناء عن نظام mesophase، وسمة أساسية من سمات وهو الزجاج الإزاحة الإيجابية حقنة 5،7. أنها تختلف الأهم فيما يتعلق تسليم الكثيرمن. يمكن الروبوتات 1 و 2 في وقت واحد الاستغناء الكثيرمن إلى 8 آبار، عمود واحد على لوحة تبلور في وقت واحد. في المقابل، روبوت 3 يوزع كل الحلول مرسب 96 وذلك في خطوة واحدة. الروبوت 2 هو الأداة الوحيدة مع نصائح الكثيرمن المتاح الاستغناء. هناك إيجابيات وسلبيات المرتبطة مختلف الصكوك التي تعتمد على تطبيق معين، لن يتم وضع هذه هنا. ويكفي ان نقول ان كل عمل لمدة ثلاث وأنتجت بلورات ليالبروتينات mbrane من قبل في طريقة المتوسط. الخطوات التالية في العملية الشاملة لتصميم الهيكل من قبل البلورات الجزيئات هي الحصاد والبرد بارد بلورات من إعداد لوحات كما هو موضح في هذه المقالة الفيديو وتسجيل وعملية حيود الأشعة السينية منها. وتغطي هذه المواضيع في مقالات منفصلة إن الرب في هذه السلسلة 1،15.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> مكونات </td></tr><tr><td> براير (الرول) </td><td> فيشر العلمية </td><td> 50820937 </td><td> أداة </td></tr><tr><td> الغاز محكم المحاقن </td><td> هاميلتون </td><td> 81030 </td><td> أداة </td></tr><tr><td> الزجاج coverslips </td><td> مارينفيلد </td><td> 01029990911 </td><td> يمكن التخلص منها </td></tr><tr><td> لوحات الزجاج </td><td> مارينفيلد </td><td> 1527127092 </td><td> يمكن التخلص منها </td></tr><tr><td> العنقاء LCP روبوت </td><td> الفن روبنز </td><td> NA </td><td> أداة </td></tr><tr><td> في المتوسط ​​الروبوت </td><td> Anachem / جيلسون </td><td> NA </td><td> أداة </td></tr><tr><td> مختبر الكمبيوتر المحمول </td><td> مختلف </td><td> NA </td><td> أداة </td></tr><tr><td> البعوض LCP روبوت </td><td> TTP LABTECH </td><td> NA </td><td> أداة </td></tr><tr><td> مثقب الشريط التبادل المزدوج عصا </td><td> سوندرز شركة (ثقب اللكم) </td><td> تخصيص </td><td> يمكن التخلص منها </td></tr><tr><td> الكثيرمن الحلول </td><td> مختلف </td><td> مختلف </td><td> كاشف </td></tr><tr><td> تنقية المياه </td><td> ميليبور </td><td> NA </td><td> كاشف </td></tr><tr><td> المطر-X </td><td> قذيفة العناية بالسيارات </td><td> 80199200 </td><td> كاشف </td></tr><tr><td> حقنة نصائح </td><td> هاميلتون </td><td> 7770-020 (قياس 22) </td><td> أداة </td></tr><tr><td> الأنسجة </td><td> مختلف </td><td> مختلف </td><td> يمكن التخلص منها </td></tr><tr><td> زجاجة ماء </td><td> مختلف </td><td> NA </td><td> كاشف </td></tr></tbody></table>

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

هيكل الوظائف الدراسات البروتينات الغشاء تستفيد كثيرا من وجود المتاحة عالية الدقة هياكل 3-D من النوع المقدمة من خلال البلورات بالأشعة السينية الجزيئات (MX). عنصر أساسي من MX هو إمداد ثابت من بلورات حيود الجودة مثالي. وأو شحمي في المتوسط ​​مكعب المرحلة (LCP) طريقة لبلورة البروتينات الغشاء هي واحدة من العديد من الطرق المتاحة لبلورة البروتينات الغشاء. فإنه يجعل من استخدام mesophase bicontinuous التي تنمو البلورات. كوسيلة من وسائل، وكان له بعض النجاحات الرائعة في الآونة الأخيرة واستقطبت الكثير من الاهتمام مع العديد من المجموعات البحثية المهتمة الآن في استخدامه. واحدة من التحديات المرتبطة الأسلوب هو أن mesophase استضافة لزج للغاية ومثبت، تذكرنا معجون أسنان سميكة. وهكذا، فإنه يدويا الاستغناء بطريقة استنساخه في كميات صغيرة في بلورة الآبار يتطلب مهارة وصبر ومطرد من ناحية. بروتوكول للقيام جوش التي وضعت في الغشاء الهيكلي والوظيفي الأحياء (MS &amp; FB) المجموعة 1-3. مقالات الفيديو إن الرب واصفا الطريقة هي 1،4 المتاحة. النهج دليل لإقامة المحاكمات في المتوسط ​​لديها مزايا واضحة مع التطبيقات المتخصصة، مثل وضوح الشمس والتحسين الاشتقاق. ومع ذلك فإنه لا يعاني من كونها وسيلة إنتاجية منخفضة. هنا، ونحن يبرهن على وجود بروتوكول للأداء في بلورة التجارب المتوسط ​​آليا. والروبوت يوفر مزايا السرعة والدقة، والدقة، والتصغير والقدرة على العمل بشكل مستمر لفترات طويلة تحت ما يمكن اعتباره ظروف عدائية كما هو الحال في الظلام، في جو خفض أو في درجات حرارة منخفضة أو عالية. يمكن للروبوت في المتوسط، عندما تستخدم بشكل صحيح، تحسن كثيرا من إنتاجية بنية البروتين الغشاء وظيفة البحث من خلال تسهيل بلورة التي تعد واحدة من الخطوات البطيئة فيوعموما خط أنابيب هيكل المصير. في هذه المقالة الفيديو، ونحن لشرح استخدام ثلاثة الروبوتات المتاحة تجاريا التي يمكن الاستغناء عن mesophase لزج ودبق جزءا لا يتجزأ من المتوسط ​​في crystallogenesis. تم تطوير أول روبوت في MS &amp; FB مجموعة 5،6. أصبحت في الآونة الأخيرة اثنين آخرين المتاحة ويتم تضمين هنا للتأكد من اكتمالها. ويقدم لمحة عامة عن بروتوكول المشمولة بهذه المادة في الشكل 1. تم تنفيذ كافة التلاعب في درجة حرارة الغرفة (~ 20 ° C) في ظل الظروف المحيطة.

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

يوصف هنا نهج الروبوتية إلى الإنتاجية العالية بلورة البروتينات الغشاء في mesophases شحمي لاستخدامها في تصميم هيكل الجزيئات باستخدام الأشعة السينية البلورات. وقدم ثلاثة روبوتات قادرة على التعامل مع لزجة لزجة والبروتين لادن mesophase جزءا لا يتجزأ من أسلوب.

استخدام روبوت لارتفاع الإنتاجية البلورة من بروتينات الغشاء في Mesophases شحمي

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

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Biology

17.4K Views.  Trinity College Dublin. يوصف هنا نهج الروبوتية إلى الإنتاجية العالية بلورة البروتينات الغشاء في mesophases شحمي لاستخدامها في تصميم هيكل الجزيئات باستخدام الأشعة السينية البلورات. وقدم ثلاثة روبوتات قادرة على التعامل مع لزجة لزجة والبروتين لادن mesophase جزءا لا يتجزأ من أسلوب.

Video: 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.

بلورة البروتينات الغشائية لتحديد الهيكل باستخدام Mesophases Lipidic

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.

تقييم المحاكمات تبلور ثنائي الأبعاد للبروتينات غشاء الصغيرة للدراسات البيولوجيا الهيكلية التي البلوريات الكترون

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.

البلورة البروتين لالبلورات بالأشعة السينية

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.

بلورة البروتينات في الغشاء Mesophases Lipidic

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.

الإنتاجية العالية للبروتينات غشاء البلورة باستخدام أسلوب Bicelle Lipidic

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.

الحصاد والبرد تبريد بلورات من بروتينات الغشاء يزرع في Mesophases شحمي لتحديد هيكل للعلم البلورات ضخم الجزيئات

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.

عالية الإنتاجية تنقية تقارب الموسومة البروتينات المؤتلف

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.

الكيمياء الحيوية والعليا تقييم مجهرية مرت من كتلة الدهون في<em&gt; Caenorhabditis ايليجانس</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.

وهناك طريقة عالية الإنتاجية لقياس معدل الترشيح الكبيبي في الفئران وإدراكا

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.

عالية الإنتاجية إبر دقيقة جدا لقنفذ البحر بيضات ملقحة

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 ...