المؤلفين أشكر الدكتور كينت سيلي، مدير مركز ديسكفري المخدرات والابتكار (CDDI) مرفق البروتيوميات الأساسية في جامعة جنوب فلوريدا لاستخدام هذا المرفق. وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح / NIDCD R01 DC004295 إلى BHAS

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الكتاب تعلن أي تضارب مصالح.

<html> الخطوات الرئيسية لتحقيق أقصى قدر من تحديد البروتين من ظهارة حسية القوقعة هي: 1) استخدام endoproteinases متعددة لعملية الهضم، 2) استخدام تقنيات فصل متعددة، و 3) استخدام مطياف الكتلة عالية الدقة. تطبيق الانزيمات متعددة يزيد من عدد من الببتيدات ويحسن التغطية تسلسل البروتين، ?...

البروتينات هي دراسة النظم البيولوجية المعقدة من خلال تحليل التعبير البروتين، وظيفة، والتعديلات، والتفاعلات 1. وقد تم استخدام عدة طرق للتحليل بروتيوم في الأذن الداخلية، بما في ذلك الأجسام المضادة ميكروأري 2، ثنائي الأبعاد الكهربائي للهلام 3-5، وDIGE 6. ومع ذلك، فقد تم التعرف فقط على عدد محدود من البروتينات وتتميز 2،7-10، مقارنة مع أكثر من 10،000 الجينات والعلامات تسلسل أعرب (التكنولوجيات السليمة بيئيا) التي تم تحديدها في الأذن الداخلية 11،12، MS هو الأسلوب الأكثر شيوعا شاملة و في البروتيوميات لتوصيف البروتين. تحليل عينات البروتين المعقدة، مثل القوقعة، يمكن أن يكون تحديا. ومع ذلك، فإن الجمع بين تقنيات فصل متعددة مع MS تمكن من تحديد عدد أكبر من الببتيدات والبروتينات، ويرجع ذلك إلى زيادة تركيز مجموعة ديناميكية وقدرة الذروة 13. chromatogra متعددة الأبعادفيز البروتين يقلل من مخاليط معقدة للغاية من خلال السماح للاستخدام آليات امتصاص مختلفة. هناك نوعان من يشيع استخدام MS نهج تحليل بروتيوم، بندقية والبروتينات من أسفل إلى أعلى. في البروتيوميات بندقية، وهي مزيج من البروتينات سليمة يتم هضم إنزيمي وفصلها باستخدام اللوني المتعدد الأبعاد مع القوي الموجبة لتبادل اللوني (SCX)، يليه اللوني السائل عكس المرحلة (RPLC) 14،15. تخضع الببتيدات فصلها لبالترادف MS وقاعدة بيانات البحث 15. والميزة الرئيسية لهذه التقنية هو أن الآلاف من البروتينات يمكن تحديدها في تحليل واحد والأسلوب هو أكثر ملاءمة للبروتينات الغشاء. في نهج من أسفل إلى أعلى، ويتم فصل خليط من البروتين، وعادة عن طريق واحد أو الكهربائي ثنائي الأبعاد، والعصابات البروتين الفردية أو بقع قطع ويتفاعل مع انزيم مثل التربسين، مما أدى عادة في الببتيدات المتعددة. ومع ذلك، وآخر أكثر حداثةوضعت لاي نهج الكهربي، وتستخدم في تحليل البروتينات من أسفل إلى أعلى، هو GELFrEE. هذه التقنية fractionates عينات البروتين في السائل المرحلة ويجعلها أقل تعقيدا قبل التحليل. هذا الأسلوب هو استنساخه، ويوفر الانتعاش عالية من البروتين، ويقلل من توزيع البروتينات وفرة عالية في عينات البروتين المعقدة 16. الببتيدات الناتجة عن البروتينات فصل، يتم تحليلها من قبل MS، باستخدام الببتيد البصمات الشامل أو بالترادف MS (MS / MS)، لإنشاء علامات تسلسل لقاعدة البيانات بالبحث 17-19. بعض من أهم مزايا استخدام نهج من أسفل إلى أعلى هي القدرة على الحصول على فصل عالية الدقة والتغطية واسعة النطاق البروتين. البروتينات من أسفل إلى أعلى هو الأسلوب الأكثر استخداما على نطاق واسع في تحليل البروتينات 20، وبالتالي، تتوفر العديد من الأدوات المعلوماتية الحيوية. بالإضافة إلى ذلك، يمكن فصل البروتينات في خليط معقد قبل الهضم، وحتى لا يكون هناك فرصة أكبر لتحديد الهوية. واحدة من التحديات الرئيسيةفي استخدام الأذن الداخلية لتحليل البروتين هو صغر حجمها، وسهولة الوصول المقيدة، ونوع من الخلايا التنوع 21. بالإضافة إلى ذلك، البروتينات الرئيسية التي تميز وظائفه، مثل القنوات الأيونية، ونقل المستقبلات، هي بروتينات الغشاء، والتي يمكن أن يكون من الصعب عزل 22. وبالتالي، عينة إعداد بمساعدة فلتر (FASP) هو مفيد لتحليل البروتين من الأنسجة التي تقتصر لاستخراج البروتين والتي تتطلب المنظفات للذوبان الأغشية 23. هذه التصفية يسمح للتحليل MS من الغشاء والبروتينات القابلة للذوبان ونظرا لقدرته على عزل الببتيدات من الملوثات منخفضة الوزن الجزيئي 23،24. يصف هذا البروتوكول النهج البروتين شائعة الاستخدام التي يتم الجمع بين وتعديلها لتحليل البروتينات على حد سواء للذوبان والغشاء وزيادة عدد معرفات البروتين من الظهارة الحسية قوقعة. سنقوم بشرح استخدام البروتينات بندقية مع FASP متعددة هضم-ايون، وتبادل اللوني أيون، وارتفاع القرار MS، وتحليل البيانات. بالإضافة إلى ذلك، فإننا سوف تصف البروتينات من أسفل إلى أعلى مع GELFrEE، FASP متعددة الهضم، وارتفاع القرار MS، وتحليل البيانات.

<table border="0" cellpadding="0" cellspacing="0" width="749">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">8% Tris-acetate cartridge&#160;</td>
			<td style="width:159px;">Protein Discovery</td>
			<td style="width:133px;">42103</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>Honeywell</td>
			<td>015-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AEBSF</td>
			<td>Calbiochem</td>
			<td>101500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>AC16861</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Calbiochem</td>
			<td>616370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ASB-14&#160;</td>
			<td>Calbiochem</td>
			<td>182750-5GM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>BioRad</td>
			<td>500-0112</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">C18 column&#160;</td>
			<td>New Objective</td>
			<td>A25112</td>
			<td>75 &#956;m &#215; 10 cm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DC Protein Assay</td>
			<td>BioRad</td>
			<td>500-0116</td>
			<td>Microplate Assay Protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endoproteinase Lys-C</td>
			<td>Sigma-Aldrich</td>
			<td>P3428</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FASP Protein Digestion Kit</td>
			<td>Protein Discovery</td>
			<td>44250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid&#160;</td>
			<td>Fluka</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GELFrEE Fractionation System</td>
			<td>Protein Discovery</td>
			<td>42001</td>
			<td>GELFrEE 8100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Calbiochem</td>
			<td>108975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MacroSpin Column</td>
			<td>The Nest Group</td>
			<td>SMM SS18V</td>
			<td>Silica C18</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcystin</td>
			<td>Calbiochem</td>
			<td>475815</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Sigma-Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polysulfoethyl A Column</td>
			<td>The Nest Group</td>
			<td>202SE0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonic Dismembrator&#160;</td>
			<td>Thermo Fisher</td>
			<td>15-338-53</td>
			<td>Model 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T6567</td>
			<td>Proteomics Grade</td>
		</tr>
	</tbody>
</table>

bottom-up and shotgun proteomics to identify a comprehensive cochlear proteome

البروتيوميات هو نهج شائعة الاستخدام التي يمكن أن توفر نظرة ثاقبة النظم البيولوجية المعقدة. الظهارة الحسية قوقعة يحتوي على المستقبلات التي تنبيغ الطاقة الميكانيكية الصوتية إلى طاقة الكهربائية والكيميائية التي تتم معالجتها بواسطة الجهاز العصبي المحيطي والمركزي. وقد تم تطوير العديد من التقنيات لدراسة البروتين في الأذن الداخلية القوقعة، مثل ثنائية الأبعاد الكهربائي الفرق هلام (2D-DIGE)، الأجسام المضادة ميكروأري، ومطياف الكتلة (MS). MS هو الأداة الأكثر شمولا وتنوعا في البروتينات وبالتزامن مع أساليب الانفصال يمكن أن توفر بروتيوم متعمق لعينات بيولوجية. طرق الفصل جنبا إلى جنب مع مرض التصلب العصبي المتعدد لديه القدرة على تخصيب عينات البروتين، والكشف عن منخفض الوزن الجزيئي والبروتينات مسعور، وتحديد البروتينات وفرة منخفضة من خلال تقليل النطاق الديناميكي بروتيوم. استراتيجيات الهضم مختلفة يمكن تطبيقها على المحللة كله أو مجزأ إلى البروتين المحللة لتعزيز الببتيد والبروتيناتتغطية التسلسل. يمكن تطبيق استخدام تقنيات الفصل المختلفة، بما في ذلك تبادل قوي الموجبة (SCX)، عكس المرحلة (RP)، ومزال هلام السائل الكهربائي جزء فخ (GELFrEE) للحد من تعقيد العينة قبل التحليل MS لتحديد البروتين.

للحصول على بروتيوم الأكثر شمولا من الظهارة الحسية قوقعة، مطلوب تشريح الأنسجة سريعة قبل استخراج البروتين وإعداد العينات. اثنين من التقنيات البروتين يمكن استخدامها، وبندقية من أسفل إلى أعلى البروتينات. لتحضير عينات للتحليل البروتينات طلقات نارية، تم استخدام الإجرا?...

Watch this Scientific Journal Video about Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome at JoVE.com

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

تحليل بروتيوم من الظهارة الحسية قوقعة يمكن أن يكون تحديا نظرا لصغر حجمها ولأن البروتينات الغشاء يصعب عزل والتعرف عليها. ويمكن تحديد كل من الغشاء وبروتينات قابلة للذوبان عن طريق الجمع بين أساليب وتقنيات متعددة إعدادي فصل جنبا إلى جنب مع عالية الدقة قياس الطيف الكتلي.

من أسفل إلى أعلى وبندقية البروتيوميات من التعرف على القوقعة بروتيوم الشامل

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

bottom-up-shotgun-proteomics-to-identify-comprehensive-cochlear

Research

JoVE Journal

Biology

18.6K Views.  University of South Florida.  تحليل بروتيوم من الظهارة الحسية قوقعة يمكن أن يكون تحديا نظرا لصغر حجمها ولأن البروتينات الغشاء يصعب عزل والتعرف عليها. ويمكن تحديد كل من الغشاء وبروتينات قابلة للذوبان عن طريق الجمع بين أساليب وتقنيات متعددة إعدادي فصل جنبا إلى جنب مع عا?...

Video: Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

العزلة وتحليل الخلايا الجذعية المكونة للدم من المشيمة

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

البروتيوميات من التعرف على البروتينات التفاعل مع P2X2 قنوات الكاتيون يجند بوابات

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR كليب -- وسيلة لتحديد نطاق Transcriptome مواقع الربط من البروتينات الحمض النووي الريبي تجليد

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

الكمي من البروتينات عن طريق التخصيب الببتيد Immunoaffinity اقترن الطيف الكتلي

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

متعامد تنقية البروتين الميسر عن طريق بطاقات تقارب Bispecific الصغيرة

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

[رني الفرز لتحديد الظواهر تال للمرحلة الجنينية في C. ايليجانس</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

عزلة البروتينية ريبوسوم الوليدة منضم<em&gt; في المختبر</em&gt; لتحديد المواقع وقفة بالحركة على طول مرنا

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

إنتاج<em&gt; القيطم مداري</em&gt; البيض مقتطفات من التعرف على الرنا أنيبيب المرتبطة بها

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

أنثرالين كما مصفوفة لمصفوفة بمساعدة الليزر الامتزاز / التأين التصوير على تحويل فورييه ايون السيكلوترون الرنين مطياف الكتلة

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.