ويدعم العمل في المختبر المؤلفين من خلال منحة من المعهد الوطني للعلوم الطبية العامة (GM41628) ومنحة لمعهد نيفيز للبحوث الطبية من صندوق البحوث الطبية نيلسون هيلين في مؤسسة المجتمع كانساس سيتي الكبرى.

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تعلن الكتاب أنه ليس لديهم المصالح المالية المتنافسة.

وقد أعاق الدراسات البنيوية والوظيفية متعددة فرعية المجمعات الثدييات الكروماتين إعادة عرض من حقيقيات النوى أعلى من صعوبة إعداد كيميائيا كميات مفيدة من هذه المجمعات التي تحتوي على وحدات فرعية متحولة أو تفتقر إلى بعض مفارز تماما. هناك عدد من العقبات الفنية: أولا، كان ?...

الحفظ تطويريا المجمعات إعادة عرض الكروماتين الأسرة SNF2 هي المنظمين الرئيسيين للتنظيم الكروماتين وDNA الوصول 1. إعادة عرض هذه المجمعات تشمل دائما مثل SNF2 أتباز فرعية المركزية، التي،، يجمع في بعض الحالات مع مختلف البروتينات الإكسسوارات وتشكل فرعية متعددة، الجمعيات الكلي الجزيئية. لدراسة التفاصيل الجزيئية لعملية إعادة عرض الكروماتين تعتمد على ATP، فمن المهم أن نفهم مساهمات مجموعات فرعية معينة من الوحدات الفرعية و / أو هياكل المجال لأنشطة المجمعات. مثل هذه التحليلات تتطلب القدرة على توليد المجمعات متحولة العالية النقاء التي تفتقر إلى البروتين مفارز معينة أو هياكل المجال. وقد ركزت الدراسات السابقة هيكل وظيفة من ATP التي تعتمد على المجمعات الكروماتين إعادة عرض على نطاق واسع في النظام النموذجي الخميرة بسبب manipulability متفوقة من الجينوم الخميرة (انظر، على سبيل المثال، الحكام 1-4). نظرا لصونتكوين فرعية والوظائف بين المجمعات إعادة orthologous، قدمت دراسات لهيكل وظيفة من المجمعات إعادة الخميرة نظرة ثاقبة على نظرائهم في حقيقيات النوى أعلى. ومع ذلك، لا توجد أنواع محددة ملموسة الخلافات بين المجمعات إعادة عرض، الناتجة عن المكسب أو الخسارة من أنواع محددة مفارز، ربح أو خسارة من المجالات أنواع محددة من مفارز المحفوظة، وتقلب تسلسل ضمن مجالات الحفظ مفارز المحفوظة. من حيث المبدأ يمكن أن يكون الدافع وراء هذه الاختلافات بسبب الحاجة للخلايا حقيقية النواة أعلى على التكيف مع بيئات الجزيئية والخلوية الجديدة. وبالتالي، فإن فهم كيفية مساهمة مفارز من أعلى المجمعات إعادة حقيقية النواة لعملية إعادة النيوكليوسومات هو قيمة، لأنه يسلط الضوء ليس فقط على الآليات الأساسية لعملية إعادة عرض الكروماتين ATP-تعتمد، ولكن أيضا يمكن أن توفر معلومات قيمة حول الآليات التي بنية الكروماتين والتعبير الجيني في اثوينظم حقيقيات النوى لها. حتى الآن، كانت هناك فقط تقتصر الدراسات البنيوية والوظيفية متعددة فرعية-الثدييات المجمعات إعادة عرض الكروماتين، ويرجع ذلك جزئيا إلى الصعوبات في الحصول على تعريفها كيميائيا الكروماتين المجمعات إعادة عرض وsubcomplexes. لقد التحايل جزئيا على هذه الصعوبات مع الإجراءات الموضحة أدناه، والذي يستخدم في تنقية immunoaffinity لإعداد سليمة INO80 أو INO80 subcomplexes من خلايا الإنسان التعبير عن ثابت N-عضال FLAG حاتمة الموسومة النوع البري أو الإصدارات متحولة من Ino80 5-7 (الشكل 1) . للحصول على المجمعات INO80 سليمة من الخلايا البشرية، ويستخدم FLP بوساطة إعادة التركيب لتوليد خطوط الخلايا المعدلة وراثيا HEK293 التعبير عن ثابت حاتمة FLAG cDNAs الموسومة ترميز مفارز مجمع INO80 8-10. لأن الإفراط في التعبير عن مفارز INO80 يمكن أن تكون سامة إلى حد ما، فمن الضروري عزل والحفاظ على خطوط الخلايا نسيلي تحت شارك انتقائيةnditions لضمان استقرار التعبير التحوير خلال العديد من الممرات اللازمة للتوسع في مزارع الخلايا على نطاق واسع. للحصول على subcomplexes INO80 أصغر التي تحتوي على مجموعة فرعية فقط من الوحدات الفرعية، وقد استخدمنا بنجاح النهجين (الشكل 2A، B). في البداية، وتوليد HEK293 FLP-في خطوط الخلايا معربا عن ستابلي الإصدارات متحولة من Ino80 التي تفتقر المجالات المطلوبة للتفاعل مع وحدات فرعية محددة 5. بدلا من ذلك، يستخدم ضربة قاضية سيرنا بوساطة في استنزاف الوحيدات المطلوب من خلايا إبداء الموسومة FLAG INO80 فرعية المناسبة (بيانات غير منشورة). أخيرا، لتنقية المجمعات INO80 الإنسان، يتم استخدام FLAG اللوني القائم الاغاروز 11 إلى إثراء جزء التي تحتوي على مقتطفات من INO80 النووية، وبالتالي الحد من فعالية وجود تلوث البروتينات عصاري خلوي في جزء نهائي يتضمن النقي INO80 أو INO80 subcomplexes.

<table border="0" cellpadding="0" cellspacing="0" width="890">
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			<td height="21" style="height:21px;width:359px;">Name of Reagent/Material</td>
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			<td style="width:147px;">Catalog Number</td>
			<td style="width:173px;">Comments</td>
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			<td>Life Technologies</td>
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			<td>Life Technologies</td>
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			<td height="21" style="height:21px;width:359px;">pOG44 Flp-Recombinase Expression Vector</td>
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			<td>SAFC</td>
			<td style="width:147px;">12138C</td>
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			<td height="21" style="height:21px;">TARGETplus SMARTsiRNA pool</td>
			<td>Dharmacon / Thermo Scientific</td>
			<td style="width:147px;">various</td>
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			<td>Dharmacon / Thermo Scientific</td>
			<td style="width:147px;">#B-002000-UB-100</td>
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			<td height="21" style="height:21px;">Lipofectamine RNAiMAX&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">13778</td>
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			<td height="21" style="height:21px;width:359px;">Opti-MEM Reduced Serum Medium&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">51985-091</td>
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			<td height="21" style="height:21px;">1x FLAG Peptide</td>
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			<td height="21" style="height:21px;">Micro Bio-Spin Chromatography Column</td>
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			<td height="21" style="height:21px;width:359px;">Anti-FLAG M2 antibody, mouse</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F3165</td>
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			<td height="21" style="height:21px;width:359px;">Anti-FLAG M2 antibody, rabbit</td>
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			<td style="width:173px;"></td>
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			<td>Beckman-Coulter</td>
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			<td style="width:173px;"></td>
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			<td style="width:147px;">353348</td>
			<td style="width:173px;"></td>
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			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
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		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:173px;"></td>
		</tr>
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generation and purification of human ino80 chromatin remodeling complexes and subcomplexes

INO80 المجمعات الكروماتين إعادة تنظيم ديناميات النيوكليوسومات والحمض النووي الوصول عن طريق حفز ATP التي تعتمد على إعادة جسيم نووي. تتكون المجمعات INO80 الإنسان من 14 مفارز البروتين بما في ذلك Ino80، وهو SNF2 مثل أتباز، الذي يخدم على حد سواء كما الوحيدات الحفازة وسقالة لتجميع المجمعات. مهام مفارز الأخرى والآليات التي تساهم في إعادة النشاط لونين مجمع INO80 وتبقى غير مفهومة، ويرجع ذلك جزئيا إلى التحدي المتمثل في توليد التجميع الثانوي INO80 في الخلايا البشرية أو أنظمة تعبير مغايرة. يصف هذا البروتوكول إن الرب إجراء من شأنه أن يسمح تنقية INO80 الكروماتين subcomplexes إعادة البشري التي تفتقر حدة فرعية أو مجموعة فرعية من الوحدات الفرعية. الموسومة N-عضال حاتمة FLAG يتم تقديمها Ino80 [كدنا مستقر في الكلى الجنينية البشرية (HEK) 293 خطوط الخلايا باستخدام FLP بوساطة إعادة التركيب. في حالة عدم وجود فرعية من مفارز مجمع INO80 هي بالبريد حذفها، واحد عن البروتينات Ino80 بدلا متحولة التي تفتقر إلى منصة اللازمة لتجميع تلك الوحدات الصغرى. في حالة وحدة فرعية الفردية هي أن تنفد، واحدة transfects الرناوات siRNAs تستهدف هذه الوحدة الفرعية إلى خط الخلية HEK 293 التعبير عن ثابت FLAG الموسومة Ino80 أتباز. يتم إعداد مقتطفات النووية، ويتم تنفيذ FLAG مناعي لإثراء الكسور البروتين تحتوي على مشتقات Ino80. ويمكن بعد ذلك يتم تحليل التراكيب من subcomplexes INO80 تنقيته باستخدام أساليب مثل immunoblotting والفضة تلطيخ، ومطياف الكتلة. وINO80 وINO80 subcomplexes المولدة وفقا لهذا البروتوكول يمكن تحليلها باستخدام مختلف المقايسات الحيوية، والتي تم وصفها في البروتوكول إن الرب المصاحب. الأساليب المذكورة هنا يمكن تكييفها للدراسات من الخصائص الهيكلية والوظيفية في أي الثدييات فرعية متعددة الكروماتين إعادة عرض والمجمعات تعديل.

ويبين الشكل 1 مخطط تدفق يلخص الإجراءات المستخدمة لتوليد وتنقية، وتميز INO80 ATP التي تعتمد على المجمعات إعادة عرض لونين الإنسان. كما هو موضح في الشكلين 2 و 3، وتمكن هذه الإجراءات توليد كلا نوع INO80 وINO80 subcomplexes...

Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

يصف هذا البروتوكول إجراء لتوليد وتنقية النوع البري والإصدارات متحولة من مجمع INO80 إعادة عرض الكروماتين البشري. حاتمة الموسومة يتم التعبير عن إصدارات مفارز INO80 مستقر في خلايا HEK293، ويتم تنقية المجمعات والمجمعات كاملة تفتقر إلى مجموعات محددة من قبل مفارز immunoaffinity اللوني.

جيل وتنقية الإنسان INO80 ونين إعادة عرض مجمعات وSubcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

generation-purification-human-ino80-chromatin-remodeling-complexes

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9.7K Views.  Stowers Institute for Medical Research.  يصف هذا البروتوكول إجراء لتوليد وتنقية النوع البري والإصدارات متحولة من مجمع INO80 إعادة عرض الكروماتين البشري. حاتمة الموسومة يتم التعبير عن إصدارات مفارز INO80 مستقر في خلايا HEK293، ويتم تنقية المجمعات والمجمعات كاملة تفتقر إلى مجموعات محددة ...

Video: Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

تنقية والتخيل من الأنفلونزا من المجمعات بروتين نووي ريبوزي الفيروسية

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

الإنسان عزل جزيرة البنكرياس : الجزء الثاني : تنقية الثقافة والإنسان من الفشوت

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

تلطيخ من البروتينات في الهلام مع Coomassie G - 250 دون عضوي حمض الخليك والمذيبات

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

تصور من خلايا خلالية لشبكة (ICC) في الفئران كاجال

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

تحضير وتنقية وتوصيف انثانيد المجمعات للاستخدام وكلاء والتباين للتصوير بالرنين المغناطيسي

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

لونين عزل من قبل تنقية الحمض النووي الريبي (السقسقة)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

جيل وتنقية، وتوصيف الأنواع الغازية خلية البروتين DISC1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

جيل من جودة عالية لونين القالب الحمض النووي مناعي لتسلسل عالية الإنتاجية (رقاقة وما يليها)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

كفاءة الإنتاج وتنقية المؤتلف الفئران Kindlin-3 من خلايا الحشرات للدراسات البيوفيزيائية

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

فحوصات البيوكيميائية لتحليل أنشطة ATP التي تعتمد على لونين إعادة عرض الانزيمات

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...