LRC - V. يرغب في تكريس هذه الورقة لذكرى الدكتور جونسون Adriel دال ، وهو أستاذ الرائع الذي الاولوية رقم واحد كان دائما في تعليم الطلاب. نحن نفتقدك ، Adriel. نحن ممتنون لمورينو جاكلين لمساعدتها في أداء والتجارب المذكورة في هذه الدراسة. وأيد هذه الدراسة عن طريق منحة تقدم إلى CY من مؤسسة العلوم الوطنية ، MCB - 0615390 ، وعبر صناديق المبتدئة التي قدمت إلى الصليب الأحمر اللبناني - V من جامعة الاباما في هانتسفيل.

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Bacteriol. 183, 6898-6907 (2001).</li><li>Gaynor, M., Mankin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrolide+antibiotics:+binding+site,+mechanism+of+action,+resistance.">Macrolide antibiotics: binding site, mechanism of action, resistance.</a> Curr. Top. Med. Chem. 3, 949-961 (2003).</li><li>Polacek, N., Mankin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosomal+peptidyl+transferase+center:+structure,+function,+evolution,+inhibition.">The ribosomal peptidyl transferase center: structure, function, evolution, inhibition.</a> Crit. Rev. Biochem. Mol. Biol. 40, 285-311 (2005).</li><li>Steitz, T. 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Biol. 22, 8562-8570 (2002).</li><li>Nakatogawa, H., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosomal+exit+tunnel+functions+as+a+discriminating+gate.">The ribosomal exit tunnel functions as a discriminating gate.</a> Cell. 108, 629-636 (2002).</li><li>Onouchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nascent+peptide-mediated+translation+elongation+arrest+coupled+with+mRNA+degradation+in+the+CGS1+gene+of+Arabidopsis.">Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis.</a> Genes Dev. 19, 1799-1810 (2005).</li><li>Raney, A., Law, G. L., Mize, G. J., Morris, D. 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R., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+Residues+Asp16+and+Pro24+of+tnaC-tRNAPro+Participate+in+Tryptophan+Induction+of+tna+Operon+Expression.">Conserved Residues Asp16 and Pro24 of tnaC-tRNAPro Participate in Tryptophan Induction of tna Operon Expression.</a> J. Bacteriol. 190, 4791-4797 (2008).</li><li>Cruz-Vera, L. R., Yang, R., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tryptophan+inhibits+Proteus+vulgaris+tnaC+leader+peptide+elongation,+activating+tna+operon+expression.">Tryptophan inhibits Proteus vulgaris tnaC leader peptide elongation, activating tna operon expression.</a> J Bacteriol. 191, 7001-7006 (2009).</li><li>Cruz-Vera, L. 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F. l. u. o. r. e. s. c. e. n. c. e. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=isotope-+or+biotin-labeling+of+the+5+'-end+of+single-stranded+DNA/RNA+using+T4+RNA+ligase.">isotope- or biotin-labeling of the 5 '-end of single-stranded DNA/RNA using T4 RNA ligase.</a> Nucleic Acids Res. 25, 3747-3748 (1997).</li><li>Nishigaki, K., Taguchi, K., Kinoshita, Y., Aita, T., Husimi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-ligation:+an+efficient+method+for+ligating+single-stranded+DNAs+and+RNAs+with+T4+RNA+ligase.">Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase.</a> Mol Divers. 4, 187-190 (1998).</li></ol>

الإعلان عن أي تضارب في المصالح.

ووصف هذا الإجراء عزلة في هذا التقرير هو تكرار للغاية. للأسف ، لا يمكن وجود قصيرة ببتيديل - tRNAs المنتجة من نفس تسلسل ترجم يمكن تجنبها بسهولة ، مثل ببتيديل - tRNAs قد تتوافق مع 15-20 ٪ من مجموع المواد المعزولة (الشكل 2C). قد تكون هذه الزيادة في تركيز الملوثات عندما استخدمت تركيزات أعلى من مرنا البيوتين المسمى أو عند استخراج خالية من الخلايا المستخدمة قديمة أو أعيد استخدامها عدة مرات. لذا ، فمن المهم جدا للتحكم في جودة وتركيز عناصر في التفاعلات الترجمة المختبر. بدلا من ذلك ، أعادت عالية الكفاءة التجارية خالية من الخلايا مقتطفات المتاحة التي يمكن استخدامها للأغراض نفسها (نظام PURE) 18. باستخدام هذا الأسلوب ، يتم تضمينها بشكل عشوائي البيوتين - 16 - UMP على طول ومرنا الهدف. هناك احتمال أن بعض ORFs تحتوي على مساحات يوريدين قد أدرجت هذا المعدل في متواليات النوكليوتيدات بهم ، مما يؤثر على ترجمتها. تركيزات النسبية متغير biotin-16-UTP/UTP أن يتم اختبارها خلال تجميعي للمرنا من أجل الحصول على ترجمة أكثر كفاءة. ويمكن استخدام أساليب بديلة لوضع العلامات البيوتين ، واستهداف 5&#39; نهاية ، ونهاية الأكثر استقرارا من mRNAs. يمكن (ط) أن يرفق البيوتين إلى نهاية 5&#39; - مرنا للاستخدام 3&#39; - NH2ATP (3&#39; الأمينية ، 3&#39; - deoxyadenosine ثلاثي الفوسفات) و T4 RNA يغاز 19. يمكن (ب) أن يرفق 5&#39; نهاية البيوتين المسمى قليل النوكليوتيد إلى نهاية 5&#39; - مرنا للاستخدام الحمض النووي الريبي T4 يغاز كذلك 20. يمكن (الثالث) كما في نهاية 3&#39; البيوتين المسمى oligodeoxynucleotides التي تتوافق مع تسلسل تكميلية في نهاية 5&#39; - مرنا من أن تستخدم لعزل المجمعات ترجمة. يمكن أن تعلق هذه oligodeoxynucleotides البيوتين المسمى إلى الشركات الصغيرة والمتوسطة قبل العزلة. في وقت لاحق SMB تعلق على البيوتين المسمى oligodeoxynucleotides يمكن أن تكون مختلطة مع الترجمة خليط التفاعل للسماح لهم التفاعل مع تسلسل مرنا التكميلية. بعد العزلة ، والهجين RNA - DNA المنطقة -- والمنطقة التهجين بين oligodeoxynucleotide البيوتين المسمى وتسلسل مرنا -- يمكن استخدام المتدهورة ريبونوكلياز H ، التي تفصل بين المجمعات المتعثرة من الخرز streptavidin. هذا الأسلوب قد بديلة تسمح لاستخدامها في المجمعات في التحليلات الهيكلية في المستقبل باستخدام أساليب دقة أعلى.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Tris base</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>252859</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium acetate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63049</td>
<td>&nbsp;</td>
<tr>
<td>Potassium glutamate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium acetate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>09688</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protein A sepharose 4B slurry beads</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9424</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L9783</td>
<td>&nbsp;</td>
<tr>
<td>Taq-DNA polymerase</td>
<td>&nbsp;</td>
<td>Stratagene</td>
<td>201223</td>
<td>&nbsp;</td>
<tr>
<td>T7 Maxiprep kit</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>P1300</td>
<td>&nbsp;</td>
<tr>
<td>SMB</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>Z5482</td>
<td>&nbsp;</td>
<tr>
<td>Biotin-16-UTP</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1388908</td>
<td>&nbsp;</td>
<tr>
<td>ATP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>GTP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8877</td>
<td>&nbsp;</td>
<tr>
<td>PEP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7002</td>
<td>&nbsp;</td>
<tr>
<td>PK</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7768</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylenglycol 8000</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>81268</td>
<td>&nbsp;</td>
<tr>
<td>Folinic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>47612</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>85558</td>
<td>&nbsp;</td>
<tr>
<td>tRNA from E. coli</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R1753</td>
<td>&nbsp;</td>
<tr>
<td>DEPC</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5758</td>
<td>&nbsp;</td>
<tr>
<td>Tricine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5816</td>
<td>&nbsp;</td>
<tr>
<td>L-amino acids</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>LAA21</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>43815</td>
<td>&nbsp;</td>
<tr>
<td>magnetic separation stands</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>Z5342</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of translating ribosomes containing peptidyl-trnas for functional and structural analyses

مؤخرا ، لم مفصلة الدراسات البنيوية والبيوكيميائية العديد من الأحداث الجزيئية التي تحدث في الريبوسوم خلال تثبيط تخليق البروتين بواسطة المضادات الحيوية ، وخلال التوليف ببتيد الوليدة. بعض هذه المضادات الحيوية ، والبروتينية الوليدة التنظيمية معظمها في شكل ببتيديل - tRNAs ، إما تمنع تشكيل ببتيد السندات أو إنهاء الترجمة 1-7. ووصف ظاهرة المثبطة هذه الأحداث يمكن أن يوقف حركة الريبوسوم &quot;اعتقال متعدية&quot;. وقد تبين اعتقال الترجمة الناجمة عن المضادات الحيوية an إما أو ببتيد الوليدة لتنظيم التعبير عن جينات لها دور في الوظائف الخلوية متنوعة مثل نمو الخلايا ، ومقاومة المضادات الحيوية ، إزفاء البروتين واستقلاب الخلية 8-13. معرفة كيفية المضادات الحيوية والبروتينية الوليدة يغير وظيفة تنظيمية الريبوسوم أمر ضروري إذا أردنا أن نفهم الدور الكامل للالريبوسوم في الترجمة ، في كل حي. هنا ، نحن تصف منهجية بسيطة التي يمكن استخدامها لتنقية ، حصرا ، للتحليل ، تلك ريبوسوم ترجمة ومرنا محددة ومعينة تحتوي على الحمض الريبي النووي النقال ببتيديل - 14. ويستند هذا الإجراء على العزلة الانتقائي للريبوسوم ترجمة منضم إلى مرنا البيوتين المسمى. وتفصل هذه المجمعات متعدية من ريبوسوم أخرى في الخليط نفسه ، وذلك باستخدام الخرز ممغطس streptavidin (SMB) ، والمجال المغناطيسي (وسط). وقد تم تجميع mRNAs البيوتين المسمى النسخ بواسطة فحوصات الاعادة باستخدام القوالب وشظايا الحمض النووي لتوليد PCR التي تحتوي على المروجين T7 النسخي. T7 بوليميريز RNA تتضمن البيوتين - 16 - UMP من البيوتين UTP ، في ظل ظروفنا وتدمج ما يقرب من عشرة البيوتين - 16 - UMP الجزيئات في الإقليم الشمالي 600 مرنا مع الحزب الحاكم على نسبة 25 ٪. ثم يتم عزل هذه البيوتين المسمى mRNAs ، واستخدامها في المختبر في إجراء فحوصات الترجمة مع عامل الإصدار 2 (RF2) - المنضب خالية من الخلايا التي تم الحصول عليها مقتطفات من سلالات كولاي التي تحتوي على نوع البرية أو ريبوسوم متحولة. وتعثرت ريبوسوم ترجمة البيوتين المسمى متواليات مرنا في المنطقة رامزة توقف ، ونظرا لغياب بروتين RF2 ، الذي يحقق في العادة إنهاء الترجمة. معزولون ريبوسوم المتعثرة التي تحتوي على توليف حديثا ببتيديل - الحمض الريبي النووي النقال وإزالتها من ردود الفعل والترجمة باستخدام SMB MF ملف. هذه الخرز فقط ربط البيوتين المحتوية على الرسائل. يمكن استخدام معزولة والمجمعات متعدية ، وتحليل الخصائص الهيكلية والوظيفية من النوع البري أو مكونات الريباسي متحولة ، أو تسلسل الحمض الريبي النووي النقال ببتيديل ، وكذلك تحديد الريبوسوم التفاعل مع المضادات الحيوية أو غيرها من العوامل الجزيئية 1،14-16. لفحص وظيفة هذه المجمعات الريبوسوم معزولة ، يمكن إجراء فحوصات ببتيديل - ترانسفيراز في وجود المضادات الحيوية بوروميسين 1. لدراسة التغيرات الهيكلية في المجمعات متعدية ، يمكن استخدام إجراءات راسخة ، مثل ط) يشابك إلى الأحماض الأمينية المحددة 14 و / أو الثاني) المقايسات حماية ألكلة 1،14،17.

Watch this Scientific Journal Video about Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses at JoVE.com

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

وعائقا رئيسيا أمام التحليلات الكيميائية الحيوية للريبوسوم تحتوي الوليدة ببتيديل - tRNAs تم بحضور ريبوسوم أخرى في نفس العينات ، ريبوسوم لم يشارك في الترجمة من تسلسل مرنا محددة يجري تحليلها. وضعنا منهجية بسيطة لتنقية ، حصرا ، على ريبوسوم تحتوي على الحمض الريبي النووي النقال - ببتيديل الوليدة من الفائدة.

عزلة ريبوسوم ترجمة المتضمن ببتيديل - tRNAs للتحليلات الوظيفية والهيكلية

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

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Research

JoVE Journal

Biology

19.7K Views.  University of Alabama Huntsville. وعائقا رئيسيا أمام التحليلات الكيميائية الحيوية للريبوسوم تحتوي الوليدة ببتيديل - tRNAs تم بحضور ريبوسوم أخرى في نفس العينات ، ريبوسوم لم يشارك في الترجمة من تسلسل مرنا محددة يجري تحليلها. وضعنا منهجية بسيطة لتنقية ، حصرا ، على ريبوسوم تحتوي على الحمض الريبي النو?...

Video: Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

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 ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

نزع الأحشاء من الشبكية والسائل الزجاجي ماوس للتحليلات البروتين

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

عزل وتوصيف المحتوية على الحمض النووي الريبي Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

الكروماتوغرافي تنقية ريبوسوم الخميرة نشطة للغاية

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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

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

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

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 ...

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 ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

العزلة والثقافة، وتوصيف وظيفي من الكبار ماوس Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

إعادة تشكيل قناة كيلو فولت في الأغشية الدهنية للدراسات الهيكلية والوظيفية

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

العزلة والتحليل الوظيفي من الميتوكوندريا من الخلايا المستزرعة وماوس الأنسجة

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.