المؤلفان بالشكر إلى أعضاء الفريق لاستعراضها شارون حرجة ، ومساهماتها في المخطوطة. ونحن ممتنون للدعم البرامج وموراشا Bikura ، علم إسرائيل مؤسسة (المنحة رقم 378/08 و1823-1807) ، وجوزيف كوهن مينيرفا مركز للبحوث غشاء حيوي ، وبرنامج الأسرة Chais زملاء للعلماء الجديد ، إبراهيم وسونيا Rochlin المؤسسة ؛ تراست الخيرية الأسرة وولفسون ، وهيلين وميلتون ألف مركز للهيكل Kimmelman الجزيئية البيولوجية والجمعية ، وتركة شلومو وBeirzwinsky سابين ؛ Meil ​​دي بوتون Aynsley ، وكارين سيم ، المملكة المتحدة.

<ol>
	<li>Ruotolo, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+mobility-mass+spectrometry+analysis+of+large+proteincomplexes.">Ion mobility-mass spectrometry analysis of large proteincomplexes.</a> Nat Protoc. 3 (7), 1139-1152 (2008).</li><li>Scarff, C. A., Thalassinos, K., Hilton, G. R., Scrivens, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Travelling+wave+ion+mobility+mass+spectrometry+studies+of+protein+structure:+biological+significance+and+comparison+with+X-ray+crystallography+and+nuclear+magnetic+resonance+spectroscopy+measurements.">Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements.</a> Rapid Commun Mass Spectrom. 22 (20), 3297-3304 (2008).</li><li>Smith, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+drift+time+measurements+from+travelling+wave+ion+mobility+spectrometry-mass+spectrometry+studies.">Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies.</a> Eur J Mass Spectrom (Chichester, Eng). 15 (2), 113-130 (2009).</li><li>Leary, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+measuring+conformation+of+solvent-disrupted+protein+subunits+using+T-WAVE+ion+mobility+MS:+an+investigation+into+eukaryotic+initiation+factors.">Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: an investigation into eukaryotic initiation factors.</a> J Am Soc Mass Spectrom. 20 (9), 1699-1706 (2009).</li><li>Lorenzen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+stoichiometry+and+conformational+changes+in+the+first+step+of+the+P22+tail+assembly.">Determination of stoichiometry and conformational changes in the first step of the P22 tail assembly.</a> J Mol Biol. 379 (2), 385-396 (2008).</li><li>Pukala, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+architecture+of+multiprotein+assemblies+determined+using+restraints+from+gas-phase+measurements.">Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements.</a> Structure. 17 (9), 1235-1243 (2009).</li><li>van Duijn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperonin+complexes+monitored+by+ion+mobility+mass+spectrometry.">Chaperonin complexes monitored by ion mobility mass spectrometry.</a> J Am Chem Soc. 131 (4), 1452-1459 (2009).</li><li>Ruotolo, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+macromolecular+protein+rings+in+the+absence+of+bulk+water.">Evidence for macromolecular protein rings in the absence of bulk water.</a> Science. 310 (5754), 1658-1661 (2005).</li><li>Ruotolo, B. T., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspects+of+native+proteins+are+retained+in+vacuum.">Aspects of native proteins are retained in vacuum.</a> Curr Opin Chem Biol. 10 (5), 402-408 (2006).</li><li>Giles, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+a+travelling+wave-based+radio-frequency-only+stacked+ring+ion+guide.">Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.</a> Rapid Commun Mass Spectrom. 18 (20), 2401-2414 (2004).</li><li>McKay, A. R., Ruotolo, B. T., Ilag, L. L., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+measurements+of+increased+accuracy+resolve+heterogeneous+populations+of+intact+ribosomes.">Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes.</a> J Am Chem Soc. 128 (35), 11433-11442 (2006).</li><li>Ruotolo, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+mobility-mass+spectrometry+reveals+long-lived,+unfolded+intermediates+in+the+dissociation+of+protein+complexes.">Ion mobility-mass spectrometry reveals long-lived, unfolded intermediates in the dissociation of protein complexes.</a> Angew Chem Int Ed Engl. 46 (42), 8001-8004 (2007).</li><li>Morton, V. L., Stockley, P. G., Stonehouse, N. J., Ashcroft, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+virus+capsid+assembly+from+non-covalent+mass+spectrometry.">Insights into virus capsid assembly from non-covalent mass spectrometry.</a> Mass Spectrom Rev. 27 (6), 575-595 (2008).</li><li>Valentine, S. J., Counterman, A. E., Clemmer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+database+of+660+peptide+ion+cross+sections:+use+of+intrinsic+size+parameters+for+bona+fide+predictions+of+cross+sections.">A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections.</a> J Am Soc Mass Spectrom. 10 (11), 1188-1211 (1999).</li><li>Mesleh, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+information+from+ion+mobility+measurements:+effects+of+the+long-range+potential.">Structural information from ion mobility measurements: effects of the long-range potential.</a> J Phys Chem. 100, 16082-16086 (1996).</li><li>Shvartsburg, A. A., Jarrold, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+exact+hard-spheres+scattering+model+for+the+mobilities+of+polyatomic+ions.">An exact hard-spheres scattering model for the mobilities of polyatomic ions.</a> Chem Phys Lett. 261, 86-91 (1996).</li></ol>

الإعلان عن أي تضارب في المصالح.

بروتوكول الموصوفة هنا يمكن تحديد المقطع العرضي الاصطدام من البروتينات أو مجمعات البروتين مع هيكل من ثلاثة مجهولة الأبعاد ، وذلك بهدف توفير المعلومات عن شكلها العام ، والتعبئة الوحيدات والطوبولوجيا. تحقيقا لهذه الغاية بمجرد اصطدام قيم المقطع العرضي وصفت من الضروري تحويل هذه القيم إلى التفاصيل الهيكلية. هذه العملية تتطلب جهودا إضافية التجريبية فضلا عن التحليل الحسابي ، التي تجري مناقشتها بايجاز أدناه. بادئ ذي بدء ، فمن المستحسن لتحليل البروتينات أو مجمعات البروتين مع الهياكل المعروفة. ويمكن توفير هذه القياسات لمراقبة الجودة مفيدة للمنهجية ، وسيمكن تقييم دقة المعلمات اقتناء بمقارنة النظرية والقيم المقاسة Ω. ويمكن حساب النظرية المقطعية المناطق من بنية الكريستال إحداثيات باستخدام MOBCAL 15،16 البرمجيات ، وهو مفتوح المصدر FORTRAN البرمجيات القائمة على السماح لتحرير رمز وفقا لاحتياجات التشغيل. لتشغيل مثل هذه الحسابات هو مطلوب منها لتعديل البرنامج بحيث يتم زيادة عدد الحسابات التكرارية في تنفيذ هيكل المدخلات والتي تقبل الملفات التي تحتوي على تنسيق عدد كبير من الذرات 1. وقد تم وضع استراتيجية IM - MS لتحديد ترتيبات طوبولوجي مفارز داخل الجمعيات المتعددة المكونات المقترحة مؤخرا 4،6. الأسلوب ينطوي على رصد مسارات التفكك المجالس البروتين إلى مكونات أصغر. ويتحقق هذا التفكك من خلال التكيف للظروف المرحلة تسيطر حل ، مما يثير لتوزيع subcomplexes تعكس &quot;لبنات البناء&quot; في الجمعيات. قياس متزامنة من القيم Ω المعقدة على حد سواء منتجات سليمة والتفكيك يولد القيود الهيكلية التي تستخدم بعد ذلك لحساب النماذج الطوبوغرافية للمجمعات البروتين. الافتراض الأساسي وراء هذه المنهجية هو أن تحتفظ subcomplexes لدت الأم تشبه تأكيدات ، بل والدراسات الحديثة أثبتت أن يتم الحفاظ على بنية حل للمنتجات التفكيك وإعادة ترتيب رئيسيا في أي حل أو أي من مراحل الغاز حدثت 4،6. الخطوة الأخيرة في تخصيص بنية البروتينات الرباعية إلى أيونات الغاز المرحلة المعقدة من المناسب القيم عبر الباب الاصطدام على نماذج الكمبيوتر الذي تم إنشاؤه. وتستخدم أساليب النمذجة من أجل استكشاف مختلف الترتيبات طوبولوجي ممكن من الوحدات الفرعية ويتم حسابها في القيم Ω السيليكو ومقارنة لتلك التجارب. وتستخدم حاليا سوى عدد قليل النهج الحسابية ، مثل الأسلوب الخشن الحبيبات spheretype يقارب قطره 1،8 مفارز. على العموم ، فإن هذا الحقل لا تزال في سنواتها الأولى ، ومطلوب مزيد من التطوير لجعل هذا النهج العام ، والتي تنطبق على مجموعة واسعة من المجمعات.

t-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis

ايون التنقل (IM) هو الأسلوب الذي يقيس الوقت الذي يستغرقه لايون للسفر عن طريق خلية الضغط تحت تأثير حقل كهربائي ضعيف. سرعة الأيونات التي تجتاز المنطقة الانجراف يعتمد على حجمها : أيونات كبيرة سوف تشهد أكبر عدد من الاصطدامات مع الغاز الخلفية خاملة (عادة N 2) ، وبالتالي السفر لأكثر ببطء من خلال الجهاز IM من تلك الأيونات التي تضم أصغر المقطع العرضي. بشكل عام ، في الوقت الذي يستغرقه لأيونات لترحيل على الرغم من أن المرحلة الغاز الكثيفة تفصل بينهما ، وفقا لاصطدامها المقطع العرضي (Ω). في الآونة الأخيرة ، واقترن الدردشة مع مطياف الكتلة الطيفي ، وأفرج عن السفر على الموجة (T - الموجة) Synapt ايون التنقل مطياف الكتلة (MS - IM). دمج مطياف الكتلة مع حركة الايونات تمكن بعدا جديدا لفصل العينة والتعريف ، مما أسفر عن طيف ثلاثي الأبعاد (الشامل لتوجيه الاتهام ، وكثافة ، والانجراف الوقت). هذا الأسلوب يسمح للفصل التداخل الطيفي في التناقص ، وتمكن من القرار مجمعات متجانسة مع كتلة متشابهة جدا ، أو نسب الشامل إلى تهمة ، ولكن الأوقات الانجراف مختلفة. وعلاوة على ذلك ، والقياسات الزمنية العائمة توفير طبقة هامة من المعلومات الهيكلية ، كما هو متعلق Ω الى الشكل العام وطوبولوجيا من أيون. يتم حساب العلاقة بين القيم المقاسة والانجراف الوقت Ω باستخدام منحنى المعايرة ولدت من البروتينات calibrant مع مقاطع عرضية محددة 1. قوة النهج IM - MS تكمن في قدرته على تحديد التعبئة الوحيدات والشكل العام للجمعيات في تركيزات بروتين micromolar ، و 1 شبه الفسيولوجية شروطه. العديد من الدراسات الحديثة على حد سواء IM البروتينات الفردية والمجمعات 2،3 بروتين غير التساهمية 4-9 ، تظاهر بنجاح أن يتم الاحتفاظ بنية البروتينات الرباعية في مرحلة الغاز ، وسلط الضوء على إمكانات هذا النهج في دراسة الهندسة المجالس بروتين غير معروف . هنا ، ونحن نقدم وصفا مفصلا لIMS - MS تحليل البروتين المجمعات باستخدام Synapt (الرباعي ايون التنقل من مرة والطيران) HDMS الصك (المياه المحدودة ، والتجاري الوحيد IM - MS وسيلة متاحة حاليا) 10. نحن تصف الخطوات التحسين الأساسية ، ومعايرة الاصطدام المقاطع العرضية ، وطرق لمعالجة البيانات وتفسيرها. الخطوة النهائية لبروتوكول يناقش أساليب لحساب القيم النظرية Ω. عموما ، فإن البروتوكول لا محاولة لتغطية كل جانب من جوانب IM - MS توصيف المجالس البروتين ، وإنما هدفها هو تقديم الجوانب العملية للطريقة الجديدة للباحثين في هذا المجال.

Watch this Scientific Journal Video about T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis at JoVE.com

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

ايون التنقل الكتلة الطيفي هو الغاز الناشئة في المرحلة التي تفصل بين الأيونات التكنولوجيا ، استنادا إلى اصطدامها المقطع العرضي والشامل. الأسلوب يوفر ثلاثي الأبعاد معلومات عن طوبولوجيا الشاملة وشكل مجمعات البروتين. هنا ، ونحن الخطوط العريضة الأساسية لإجراء وضع الصك والتحسين ، ومعايرة مرات الانجراف ، وتفسير البيانات.

تي موجة ايون التنقل الكتلة الطيفي : الإجراءات التجريبية الأساسية لتحليل البروتين مجمع

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

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24.5K Views.  Weizmann Institute of Science. ايون التنقل الكتلة الطيفي هو الغاز الناشئة في المرحلة التي تفصل بين الأيونات التكنولوجيا ، استنادا إلى اصطدامها المقطع العرضي والشامل. الأسلوب يوفر ثلاثي الأبعاد معلومات عن طوبولوجيا الشاملة وشكل مجمعات البروتين. هنا ، ونحن الخطوط العريضة الأساسية لإجراء وضع ...

Video: T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI تحضير العينة : الترا أسلوب طبقة رقيقة

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

Homarus Americanus فموي معدي تشريح الجهاز العصبي

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

سرطان بورياليس فموي معدي تشريح الجهاز العصبي

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

إجمالي تشريح المعدة من جراد البحر ، Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

موسع في فيفو الحيوانات الصغيرة التصوير بالرنين المغناطيسي وMRS : الإجراءات التجريبية الأساسية

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

باستخدام نظام التقطير GELFREE 8100 لتقطير الوزن الجزيئي وبناء مع مرحلة الانتعاش السائل

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

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

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

جمع الدم لتحليل الكيمياء الحيوية في الزرد الكبار

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

على البروتين MultiBac مجمع منصة الإنتاج في EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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

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

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

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

الأخضر الفلورسنت القائم على البروتين التعبير فحص غشاء البروتينات في<em&gt; الإشريكية القولونية</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...