ونحن ممتنون لارفين فرانك ، Palmour روبرتا وموظفي مختبرات العلوم السلوكية مؤسسة تقع في سانت كيتس وجزر الهند الغربية ، على دعمهم المستمر لعملنا الرئيسيات. وأيد هذا العمل من خلال منحة من مؤسسة البحوث الهشة العاشر من كندا (FXRFC) لSZ و-- المنح التي تعمل من المعاهد الكندية للأبحاث الصحية (ميلان) والهندسة ومجلس البحوث الوطني في كندا (MP).

Immunohistochemisty هي واحدة من أكثر التقنيات المستخدمة على نطاق واسع لوصف تعبير البروتين في الدماغ لمختلف النماذج الحيوانية التجريبية. فمن السهل نسبيا لإجراء إجراءات منهجية المناعى على أدمغة الفئران وغيرها من نماذج تجريبية مشتركة مع حجم المخ مماثلة. ومع ذلك ، لا يوجد عمل نشر لمعرفتنا أن يقدم وصفا شاملا لكيفية تنفيذ مثل هذه الإجراءات مناعي عبر الدماغ قرد بأكمله. فيما يلي وصفا مفصلا لكيفية إعداد والقرد الدماغ كله على نطاق واسع كشف المناعى للبروتينات المستهدفة المختلفة. وقد برز هذا العمل نتيجة للتعاون بين المساعي التجارية والأكاديمية. على هذا النحو ، والتفاصيل المتعلقة الأنسجة والتضمين sectioning تبقى معرفة الشخصية من وكالة الامن القومي. الجزء 1 : معاملة الحيوان ، وإعداد الأنسجة ويستخدم الدماغ من القرد الفرفت الكبار (القردوح aethiops) لهذا البروتوكول. تنفذ جميع الإجراءات في الامتثال للمجلس الكندي للرعاية الحيوان (CCAC) مبادئ توجيهية لاستخدام ورعاية الحيوانات في البحوث الطبية الحيوية 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هو الحيوان مخدرا عميق مع هيدروكلوريد الكيتامين (10 ملغم / كغم ، IM) ، الموت الرحيم مع جرعة زائدة من الصوديوم و(25 ملغم / كغم ، والرابع) بنتوباربيتال perfused transcardially مع PBS 0،1 M حتى exsanguinated تماما. </li><li style=";text-align:right;direction:rtl"> ويعقب ذلك من خلال حل بارافورمالدهيد 4 ٪ في برنامج تلفزيوني لمدة 5 دقائق (~ 1 ليتر). </li><li style=";text-align:right;direction:rtl"> يتم وضع الدماغ تخريجها في حلول السكروز متدرج PBS - مخزنة (10 و 20 و 30 ٪ ، في تسلسل) التي تحتوي على أزيد الصوديوم 0.02 ٪ وحافظت على 4 ˚ C حتى الدماغ المصارف إلى قاع الإناء. يتم استبدال حل كل بضعة أيام حتى الدماغ يخضع لتجميد sectioning. </li><li style=";text-align:right;direction:rtl"> يتم إرسالها إلى المخ المنتسبين علم الأعصاب (NSA ؛ نوكسفيل ، تينيسي) لإخضاعهم للملكية الخاصة MultiBrain ™ التضمين وتجميد sectioning التكنولوجيا. </li><li style=";text-align:right;direction:rtl"> وضعت سبعة معالم المحاذاة (7) في المصفوفة التضمين بحيث يمكن التوجه المقاطع بشكل صحيح (أي بين اليمين واليسار التوجه) وإعادة الانحياز بعد اكتمال تجهيز النسيجية. </li><li style=";text-align:right;direction:rtl"> يتم تصويرها رقميا كتلة المجمدة في مواجهة كتلة التي يتم جمعها من قبل كل قسم من كتلة تسلسلي. ويمكن في وقت لاحق الصور الرقمية ، وكذلك مقاطع من هذا الدماغ يمكن أن تستخدم لإعادة الإعمار 3D للخرائط التشريحية والنسيجية ، على التوالي. </li><li style=";text-align:right;direction:rtl"> المسلسل التعويم الحر أقسام الاكليلية ، في سماكة 50 ميكرون في القسم ، والتي تم جمعها من الدماغ كله. </li></ol> الجزء 2 : معالجة نسيجية ويتم اختيار الفروع في الفترة مكانية معينة (على سبيل المثال ، 500 ميكرون) ، ولأغراض تجريبية تم تجهيزها لدينا مع الأضداد التالية : البروتين الهشة العاشر التخلف العقلي (FMRP ؛ Chemicon ؛ تيميكولا ، كاليفورنيا) ، SMI32 (Sternberger Monoclonals شركة ؛ بالتيمور ، ماريلاند) ، وNeuN (Chemicon ؛ تيميكولا ، كاليفورنيا). FMRP هو بروتين حشوية التي وجدت بكثرة في الخلايا العصبية في أدمغة الناقل العادي والتقليب 2. NeuN (أنوية متعلق بالخلايا العصبية) يعترف الحمض النووي على وجه التحديد الملزم NeuN بروتين الخلايا العصبية الخاصة التي هي موجودة في معظم الخلايا العصبية ، ويتم توزيعها في نوى الخلايا العصبية ، وبعض العمليات perikarya القريبة العصبية 3. SMI - 32 هو ريتوكسيماب تعترف حاتمة غير فسفرته على البروتينات neurofilament 4. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في الفترة الفاصلة من 500 ميكرون ، وتستخدم ما يقرب من 140 في أقسام الأضداد. </li><li style=";text-align:right;direction:rtl"> تنفذ جميع حضانات ، ويغسل والخطوات تلطيخ خارجا مع الانفعالات معتدل باستخدام الأطباق والسلال تلطيخ المتخصصة (HistoTools ؛ نوكسفيل ، تينيسي). </li><li style=";text-align:right;direction:rtl"> يتم تحضين first المقاطع في محلول يحتوي على 0.1 ٪ M تريتون PBS/0.3 X - 100 (تكساس) / 5 ٪ أجش المصل العادي (NHS) لمدة 60 دقيقة من أجل خفض ملزم غير محدد من جزيئات الأجسام المضادة والناتجة تلوين الخلفية. </li><li style=";text-align:right;direction:rtl"> المقاطع علامة للخضوع FMRP immunostaining خطوة إضافية للانتعاش مستضد باستخدام مستضد الحل خلع القناع (ناقلات مختبرات ؛ بورلنجام ، كاليفورنيا) وفقا لتعليمات البائع. </li><li style=";text-align:right;direction:rtl"> يتم تحضين ثم المقاطع بين عشية وضحاها في أيام منفصلة في حلولها الضد منها تحتوي على 0.1 م PBS/0.3 ٪ TX / NHS 3 ٪ (مع عامل تخفيف من 000 ، 1 / ​​5 لFMRP وSMI - 32 و 1 / 2 ، 000 للأضداد NeuN. </li><li style=";text-align:right;direction:rtl"> في اليوم التالي ، يتم غسلها المقاطع لفترات 10 دقيقة ثلاثة في حل الغسيل (0.1 M TX PBS/0.3 ٪) تليها الحضانة لمدة 2 ساعة في درجة حرارة الغرفة في الأضداد المضادة للماوس biotinylated الثانوية التي أثيرت في الحصان (ناقلات مختبرات ؛ 1 / 1000 التخفيف في 0.1 ٪ M NHS PBS/0.3 TX / 3 ٪). </li><li style=";text-align:right;direction:rtl"> بعد مجموعة أخرى من يغسل 10 3 دقيقة ، يتم وضع المقاطع في حل أفيدين البيوتين ، مترافق معقدة بيروكسيداز الفجل (مختبرات المتجهات) لمدة 1 ساعة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> أخيرا ، وبعد مجموعة أخرى من يغسل ، التي يتعرض لها أبواب لمدة 10 دقيقة لتعزيز ديا النيكلminobenzidine (ني DAB) والتي تنتج رد فعل زرقاء داكنة وصمة عار داخل الخلايا العصبية مناعيا. </li><li style=";text-align:right;direction:rtl"> عند الانتهاء من إجراء النسيجي ، هي التي شنت على الشرائح الزجاجية الأبواب المغلفة الجيلاتين. </li><li style=";text-align:right;direction:rtl"> جميع أقسام هي الهواء المجفف بين عشية وضحاها وcoverslipped فقا للاجراءات المتبعة. </li></ol> الجزء 3 : النتائج الممثل : هذا الأسلوب ينتج ملف التعبير الكامل للبروتين الهدف من الاهتمام عبر دماغ القرد بأكمله. هنا نعرض الاكليلية ممثل المقاطع التي توفر لقطة من FMRP ، NeuN SMI32 والتعبير في دماغ القرد نفسه. <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> الشكل 1 : صحن يلطخ (A) وسلة (B) تستخدم لتجهيز دفعة واسعة النطاق من التعويم الحر أقسام للمناعي. <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> الشكل 2 : أقسام الاكليلية الممثل من دماغ القرد الفرفت الملون لFMRP (A) ، NeuN (B) والأجسام المضادة SMI32 (C).

<ol>
	<li>Olfert, E. D., Cross, B. M., McWilliam, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the Care and Use of Experimental Animals. , (1993).</li><li>Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P., Mandel, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FMR-1+protein+is+cytoplasmic,+most+abundant+in+neurons+and+appears+normal+in+carriers+of+a+fragile+X+premutation.">The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation.</a> Nat Genet. 4, 335-340 (1993).</li><li>Mullen, R. J., Buck, C. R., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuN,+a+neuronal+specific+nuclear+protein+in+vertebrates.">NeuN, a neuronal specific nuclear protein in vertebrates.</a> Development. 116, 201-211 (1992).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proc Natl Acad Sci U S A. 80, 6126-6130 (1983).</li></ol>

The authors have nothing to disclose.

هناك نوعان من الخطوات الحاسمة في هذا الإجراء التي تجعل نطاق واسع الكشف عن البروتينات في الدماغ بأكمله قرد ممكن. واحد هو بروتوكول التضمين وsectioning ، التي لا تزال المعرفة الشخصية من وكالة الامن القومي. والآخر هو استخدام الأطباق والسلال تلطيخ HistoTools التي تقدمها. هذا الأخي?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>anti-FMRP monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB2160</td>
<td>Requires antigen retrieval in fixed tissue.</td>
</tr>
<tr>
<td>anti-NeuN monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB377</td>
<td>N/A</td>
</tr>
<tr>
<td>anti-Neurofilament H monoclonal antibody</td>
<td>&nbsp;</td>
<td>Sternberger Monoclonals Inc.</td>
<td>SMI32</td>
<td>N/A</td>
</tr>
<tr>
<td>Antigen Unmasking Solution</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-3300</td>
<td>N/A</td>
</tr>
<tr>
<td>Normal horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050122</td>
<td>500 mL</td>
</tr>
<tr>
<td>Biotinylated Anti-Mouse IgG (H+L), made in horse</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>BA-2000</td>
<td>&nbsp;</td>
<tr>
<td>VECTASTAIN ABC Kit (Standard)</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>PK-4000</td>
<td>1.5 mg</td>
</tr>
<tr>
<td>Staining dish for floating sections</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10009</td>
<td>12 x 65 mm</td>
</tr>
<tr>
<td>Staining transport basket</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10012</td>
<td>large 
(fits 12 x 65 mm dish)</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>P8514B</td>
<td>N/A</td>
</tr>
<tr>
<td>DAB</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC11209-0050</td>
<td>N/A</td>
</tr>		</tbody>
		</table>
		</div>

batch immunostaining for large-scale protein detection in the whole monkey brain

المناعية (IHC) هي واحدة من التقنيات المستخدمة على نطاق واسع مختبر للكشف عن البروتينات المستهدفة<em&gt; في الموقع</em&gt;. أسئلة تتعلق نمط التعبير عن بروتين هدف عبر الدماغ بأكمله من السهل نسبيا للرد عند استخدام IHC في العقول الصغيرة ، مثل تلك التي من القوارض. ومع ذلك ، في الإجابة على الأسئلة نفسها في أدمغة كبيرة ومعقدة ، مثل تلك التي من قرود يقدم عددا من التحديات. هنا نقدم مقاربة منهجية للمناعي للبروتينات المستهدفة في المخ قرد الكبار. هذا النهج يعتمد على الأنسجة والتضمين sectioning منهجية المنتسبين علم الأعصاب (NSA) ، وكذلك الأدوات التي تم تطويرها خصيصا للتلوين ، دفعة من التعويم الحر المقاطع. انها نتيجة في تلوين موحدة لمجموعة من الفروع التي في فاصل زمني معين ، ويمثل الدماغ كله. يمكن أن يتعرض المقاطع الناتجة الملون لطائفة واسعة من الإجراءات التحليلية لقياس مستويات البروتين ، والسكان من الخلايا العصبية التي تعبر عن بروتين معين.

Watch this Scientific Journal Video about Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain at JoVE.com

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

على نطاق واسع مناعي للبروتينات المستهدفة في جميع أنحاء الدماغ الرئيسيات كله هو ممكن من خلال استخدام نسيج الرواية أساليب التضمين وsectioning جنبا إلى جنب مع استخدام أجهزة مبتكرة لتلوين مجموعة من المقاطع التعويم الحر متعددة في وقت معين.

Immunostaining دفعي للكشف عن البروتين واسعة النطاق في الدماغ القرد الجامعة

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

batch-immunostaining-for-large-scale-protein-detection-whole-monkey

Research

JoVE Journal

Biology

12.6K Views.  Montreal Neurological Institute. على نطاق واسع مناعي للبروتينات المستهدفة في جميع أنحاء الدماغ الرئيسيات كله هو ممكن من خلال استخدام نسيج الرواية أساليب التضمين وsectioning جنبا إلى جنب مع استخدام أجهزة مبتكرة لتلوين مجموعة من المقاطع التعويم الحر متعددة في وقت معين.

Video: Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

على نطاق واسع من شاشات مكتبات Metagenomic

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

اكتشاف بروتين Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

تنقية م. magneticum سلالة AMB - 1 بروتين المرتبطين Magnetosome MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

اسماك الزرد نطاق واسع ، واستنادا في الجسم الحي شاشة جزيء صغير

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium الفحص على نطاق واسع لتطبيقات SNP التنميط الجيني

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

استراتيجية للحساسية، مقياس كبيرة الكمية الايض

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

التحقيق البروتين البروتين التفاعلات في الخلايا الحية باستخدام الرنين ضوء بارد نقل الطاقة

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

أوديلاي: طريقة على نطاق واسع لالمعلمات المتعددة الكمي للنمو الخميرة

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

تحديد المنظمين رواية نتح النبات من خلال فحص التصوير الحراري على نطاق واسع في Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

إجراء سيميومتوميد ChIP-Seq للدراسات اللاجينية على نطاق واسع

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...