نود أن نشكر Quentina بيتي ، جامعة لينز ، النمسا ، للحصول على مساعدة عينية لها ، كاتارينا Strub ، جامعة جنيف ، سويسرا ، من أجل بناء hCD71 - GFP ، ودانيال Legler ، جامعة كونستانس ، سويسرا ، لGPI - GFP DAF ، بناء. وأيد هذا العمل من قبل صندوق العلوم النمساوية (FWF ؛ المشروع Y250 - B03) ومشروع الاتحاد الأفريقي لGEN - الوزارة الاتحادية النمساوية للعلوم والبحوث.

Microcontact الطبعة : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع ميدان micropattern من الطوابع PDMS باستخدام مشرط </li><li style=";text-align:right;direction:rtl"> غسل الحقل الذي يحتوي على micropattern بواسطة بيغ مع الإيثانول (100 ٪) والماء المقطر. </li><li style=";text-align:right;direction:rtl"> جافة الطابع PDMS مع النيتروجين أو غاز الارجون. </li><li style=";text-align:right;direction:rtl"> ماصة 50 ميكرولتر BSA - Cy5 الحل العمل (0.67 ملغ / مل) على الطابع PDMS بحيث يتم تغطية ميدانية micropattern كله مع الحل. احتضان لمدة 30 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> التخزين المؤقت غسل مجال micropattern بواسطة بيغ مع المؤسسة العامة لتحلية الفوسفات (PBS) والماء المقطر. </li><li style=";text-align:right;direction:rtl"> تجفيف PDMS مع النيتروجين أو غاز الأرجون. </li><li style=";text-align:right;direction:rtl"> وضع ختم PDMS تحت وزنه على منتصف ساترة الايبوكسي المغلفة واحدة واحتضان لمدة 30 دقيقة في درجة حرارة الغرفة في طبق بتري. </li><li style=";text-align:right;direction:rtl"> التسمية موقف الطابع PDMS على مساعدات من ساترة والشريط الطابع من الشريحة مع ملقط. </li><li style=";text-align:right;direction:rtl"> عصا تهجين ختم الغرفة الآمنة على الحقل microcontact المطبوعة على ساترة. التسمية يساعد على توطين وسط الميدان. </li><li style=";text-align:right;direction:rtl"> 60 ماصة العمل streptavidin ميكرولتر حل (50 ميكروغرام / مل) إلى دائرة رد الفعل واحتضان العينة لمدة 60 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> غسل العينة مع PBS 1 مل بإضافة العازلة إلى منفذ واحد للغرفة وإزالته في وقت واحد في الميناء الثاني مع مضخة. </li><li style=";text-align:right;direction:rtl"> ماصة 60 ميكرولتر العمل الضد biotinylated حل (10 ميكروغرام / مل في برنامج تلفزيوني تستكمل مع 0.1 ٪ توين 20 ؛ &quot;PBST&quot;) في دائرة رد الفعل واحتضان لمدة 60 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> غسل العينة مع PBST مل 1 و 1 في وقت لاحق PBS مل بإضافة العازلة إلى منفذ واحد للغرفة وإزالته مرة أخرى في الميناء الثاني مع مضخة. </li><li style=";text-align:right;direction:rtl"> تخزين الأسطح micropatterned في برنامج تلفزيوني في الظلام في درجة حرارة الغرفة حتى تكون جاهزة للخلايا البذر. </li></ol> حضانة الخلايا على سطح micropatterned : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو الخلايا تمسكا التقاء 50 ٪ في لوحة سم 3 زراعة الأنسجة. </li><li style=";text-align:right;direction:rtl"> فصل الخلايا معربا عن الطعم والبروتينات فريسة المصالح مع Ethylenediaminetetraacetic الحل (EDTA) وحامض 5 دقائق في جهاز الطرد المركزي 1000 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> تجاهل طاف بيليه وحل الخلية في النمو على المديين المتوسط ​​وفقا. </li><li style=";text-align:right;direction:rtl"> 5 دقائق في جهاز الطرد المركزي 1000 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> تجاهل طاف وحل بيليه خلية في المتوسط ​​النمو المناسبة. </li><li style=";text-align:right;direction:rtl"> تبادل PBS من دائرة رد الفعل على ساترة micropatterned بنسبة 60 ميكرولتر من التعليق الخلية. </li><li style=";text-align:right;direction:rtl"> إنشاء غرفة ترطيب التي تمرغ لوحة معقم في الماء المقطر ووضعه في طبق بتري لمنع العينة من تشغيل جافا. </li><li style=";text-align:right;direction:rtl"> احتضان خلايا أكثر من ليلة عند 37 درجة مئوية في جو من 5 ٪ CO 2 على السطوح micropatterned. </li><li style=";text-align:right;direction:rtl"> قبل تحليل الخلايا على المجهر ، وتبادل والمتوسطة من خلال حل هانك ق الملح مخزنة بما كا 2 + 2 + والمغنيسيوم. </li></ol> المجهر : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ساترة يوضع على جبل المناسبة ويتم فحص خلية التشكل تحت الضوء الأبيض. </li><li style=";text-align:right;direction:rtl"> يتم فحص جودة أنماط - BSA Cy5 بواسطة الإثارة في 647 نانومتر. </li><li style=";text-align:right;direction:rtl"> وتسجل Micropatterns فريسة للبروتين فلوري (GFP أو YFP) في 488 أو 514 نانومتر تحت إجمالي الداخلية الإثارة (TIR) ​​التأمل. </li></ol> ممثل النتائج : إذا كان هناك تفاعل بين البروتينات في الخلايا المستهدفة نمت على سطح micropatterned ، سوف تتبع فريسة إعادة توزيع الطعم. يمكن تصور أن يكون micropattern الناتجة عن تسمية مضان من البروتين الفريسة. الأهم الحصول على النقيض يوفر مقياسا مباشرا لقوة التفاعل (انظر الشكل 1). لذا فإن تقييم بسيطة من التفاعل من اثنين من البروتينات يصبح من الممكن -- دون الحاجة إلى مزيد من عملية قياس البيانات الأولية. <img src="/files/ftp_upload/1969/1969fig1.jpg" alt="الشكل 1" /> الشكل 1. إعادة ترتيب الطعم في غشاء الخلية الحية على القوة البلازما تفاعل مختلفة. وتظهر الصور TIR الخلايا T24 transfected مع (أ) CD71 - GFP على الأجسام المضادة CD71 ، CD4 (ب) و YFP عصاري خلوي على الأجسام المضادة CD4 و (ج) GPI - GFP DAF - CD59 على الضد microbiochips. البيانات هي سمة مميزة لقوي (أ) ، لا (ب) أو التفاعل الضعيف (ج). (أ) يرد من (Weghuber آخرون ، في الصحافة) و (ب) من (Schwarzenbacher وآخرون ، 2008).

<ol>
	<li>Brameshuber, M., Schwarzenbacher, M., Kaltenbrunner, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schuetz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reply+to+"Uncoupling+diffusion+and+binding+in+FRAP+experiments.">Reply to "Uncoupling diffusion and binding in FRAP experiments.</a> Nat. Methods. 6, 183-184 (2009).</li><li>Kim, P. W., Sun, Z. J., Blacklow, S. C., Wagner, G., Eck, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Zinc+Clasp+Structure+Tethers+Lck+to+T+Cell+Coreceptors+CD4+and+CD8.">A Zinc Clasp Structure Tethers Lck to T Cell Coreceptors CD4 and CD8.</a> Science. 301, 1725-1728 (2003).</li><li>Ramm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+screening:+a+technology+in+transition.">Image-based screening: a technology in transition.</a> Curr. Opin. Biotechnol. 16, 41-48 (2005).</li><li>Schwarzenbacher, M., Kaltenbrunner, M., Brameshuber, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+for+quantitative+analysis+of+protein-protein+interactions+in+living+cells.">Micropatterning for quantitative analysis of protein-protein interactions in living cells.</a> Nat. Methods. 5, 1053-1060 (2008).</li><li>Weghuber, J., Brameshuber, M., Sunzenauer, S., Lehner, M., Paar, C., Haselbruebler, T., Schwarzenbacher, M., Kaltenbrunner, M., Paster, W., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein-protein+interactions+in+the+live+cell+plasma+membrane+by+quantifying+prey+redistribution+upon+bait+micropatterning.">Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.</a> Methods in Enzymology. , .</li></ol>

شريط فيديو يحتوي على الصور التي تم توفيرها من قبل أوليمبوس ، مأخوذة من موقع التصوير للمختبر البحوث ، جامعة براون أو مستنسخة من (كيم وآخرون ، 2003).

شريط الفيديو المرافق يوضح طريقة للكشف عن البروتين البروتين التفاعلات في غشاء البلازما من الخلايا الحية (Schwarzenbacher وآخرون ، 2008 ؛. Brameshuber وآخرون ، 2009 ؛. Weghuber وآخرون ، تحت الطبع). من حيث المبدأ ، يمكن استخدام أي منصة المجهري TIRF القائم على نظام قراءات. فقط عندما يتم المطلوب حساسية عالية (على سبيل المثال للكشف عن الجزيئات واحد) ، سيكون مطلوبا المجاهر المتقدمة. لتحقيق أفضل النتائج على النقاط الهامة التالية أثناء عملية الإعداد تتطلب اهتماما خاصا : <ol class="lroman" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Cy5 تخزين مخزون حل في ظل الظروف المظلمة لتجنب التبييض من fluorophore وإعداد جيش صرب البوسنة ، حل Cy5 العمل الطازج قبل الطباعة microcontact. </li><li style=";text-align:right;direction:rtl"> يجب الحرص على عدم خدش micropattern على PDMS مع طرف ماصة واحتضان العينة تحت الظروف المظلمة لتجنب التبييض من fluorophore. </li><li style=";text-align:right;direction:rtl"> وضع على لوحة ساترة معقمة لمنع الالتصاق من الشريحة الزجاجية لطبق بتري. مرة واحدة الطابع PDMS انضمت إلى الشريحة الزجاجية ، لا نقله. </li><li style=";text-align:right;direction:rtl"> تجنب فقاعات الهواء في دائرة رد الفعل واحتضان العينة تحت الظروف المظلمة لتجنب التبييض من fluorophore. </li><li style=";text-align:right;direction:rtl"> لا تدع العينة الجافة لأكثر من 2 دقيقة لتجنب تشكيل بلورات الملح. </li><li style=";text-align:right;direction:rtl"> لمفرزة من خلايا لا تستخدم التربسين لأنها سوف يلتصق المجال خارج الخلية من بروتينات الغشاء حتى الآن التعبير عنها. وسوف يكون من المفيد تجنب التربسين للبروتينات مع معدل دوران منخفض لتشكيل micropatterns مباشرة بعد الحجز على الخلايا على السطح. </li><li style=";text-align:right;direction:rtl"> السيطرة على عدد من الخلايا المحتضنة في المجهر والتأكد من أن الخلايا لا تصل إلى أكثر من 30-50 ٪ التقاء. تأكد من أن تتم إزالة الخلايا المفرط على الفور نظرا لارتباط سريع للخلايا على سطح جسم المغلفة. </li><li style=";text-align:right;direction:rtl"> ويمكن استخدام الشرائح فقط مع أنماط جودة عالية Cy5 جيش صرب البوسنة لمزيد من التحليل. </li><li style=";text-align:right;direction:rtl"> التعبير المستقرة للبروتين فلوري فريسة هو الأفضل نظرا لعدد أكبر من الخلايا التي يمكن تحليلها في الفحص. </li><li style=";text-align:right;direction:rtl"> أمر لا غنى عنه TIRF كافية لتصور أنماط التكيف بسبب الخلفية عصاري خلوي. </li></ol> تقنية الزخرفة الدقيقة فرصا عدة لتحليل البروتين البروتين التفاعلات. أولا ، مع تقدير المحلية ، وتفاعلات البروتين البروتين حل مكانيا أيضا في الكشف عن التفاعلات الضعيفة أو غير المباشرة ممكنة بدون عيوب إعطاء عدد كبير من ايجابيات كاذبة أو السلبيات. وثانيا أنها تمكن الباحث لتحليل التفاعلات بين البروتينات والبروتين في غشاء البلازما من الخلايا الحية ، والتي من الصعب تحقيقه من خلال نهج البيوكيميائية مثل الشاشة 2 - الهجين. ثالثا النهج يسمح للكشف عن الطعم والفريسة التفاعلات التي يتم عن طريق التضمين التغيرات البيئية مثل درجة الحرارة ، وجود جزيئات البروتينات المختلفة أو غيرها أو التعديلات بعد متعدية. وبالتالي يسمح للمقايسة جهري الفرز من زوج التفاعل المعطى في سياق الخلايا الحية. علاوة على ذلك ، عن طريق الحد من كثافة سطح يجند التقاط أو استخدام يغاندس الأحادي التكافؤ ، وتحليل لحالة الراحة يصبح ذلك ممكنا. أخيرا ، إذا كنت تستخدم منصات كافية المسح الضوئي ، وعدد الخلايا تحليل مرتفع بما يكفي لمواكبة متطلبات إنتاجية عالية من شركات الأدوية لفحص المخدرات (رم ، 2005).

in-vivo detection of protein-protein interactions on micro-patterned surfaces

تفكيك شبكة تفاعل الجزيئات<em&gt; في الجسم الحي.</em&gt; هو المفتاح لفهم الآليات التي تنظم وظيفة الخلايا والأيض. وقد تم تطوير العديد من الخيارات المنهجية لمعالجة التفاعلات الجزيئية في الخلايا ، ولكن معظم هذه الطرق غير المباشرة تعاني من عدم وليس الكمية ، وبالتالي بالكاد. على العكس من ذلك ، أدخلت بضعة الراقية النهج الكمي ، والتي من الصعب مع ذلك توسيع لإنتاجية عالية. الجمع بين قدرات إنتاجية عالية مع إمكانية استخراج المعلومات الكمية ، ونحن وضعت مؤخرا مفهوم جديد لتحديد البروتين البروتين التفاعلات (Schwarzenbacher<em&gt; وآخرون</em&gt; ، 2008). هنا ، نحن تصف بروتوكول تفصيلية لتصميم وبناء على هذا النظام الذي يسمح لتحليل التفاعلات بين البروتينات وfluorophore المسمى (&quot;فريسة&quot;) وبروتين الغشاء (&quot;الطعم&quot;)<em&gt; في الجسم الحي.</em&gt;. وعلى سطوح الخلايا مطلي micropatterned functionalized مع أجسام مضادة ضد الطعم exoplasmic المجال. ويعاير الطعم والفريسة التفاعلات عن طريق إعادة توزيع فريسة الفلورسنت. يتميز الأسلوب حساسية عالية وصولا الى مستوى الجزيئات واحدة ، لديها القدرة على الكشف عن التفاعلات الضعيفة ، وارتفاع القدرة الإنتاجية ، مما يجعلها أداة الفحص حسب مقتضى الحال.

Watch this Scientific Journal Video about In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces at JoVE.com

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

هذا الفيديو يبين تحليل التجارب مع لاحقة من البروتين البروتين التفاعلات عن طريق استخدام الصغرى منقوشة السطوح. النهج توفر إمكانية الكشف عن تفاعلات البروتين في الخلايا الحية ، ويجمع بين قدرات إنتاجية عالية مع إمكانية استخراج المعلومات الكمية.

في الجسم الحي ، الكشف عن البروتين البروتين التفاعلات على مايكرو نقوش السطوح

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

in-vivo-detection-protein-protein-interactions-on-micro-patterned

Research

JoVE Journal

Biology

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

10.7K Views.  Johannes Kepler Universitat Linz. هذا الفيديو يبين تحليل التجارب مع لاحقة من البروتين البروتين التفاعلات عن طريق استخدام الصغرى منقوشة السطوح. النهج توفر إمكانية الكشف عن تفاعلات البروتين في الخلايا الحية ، ويجمع بين قدرات إنتاجية عالية مع إمكانية استخراج المعلومات الكمية.

Video: In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

انقسام اليوبيكويتين الخميرة يستند الغشاء الهجين ثنائي (الأسطورة) النظام : أداة قوية لتحديد البروتين البروتين التفاعلات

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

التصوير البروتين البروتين التفاعلات في الجسم الحي</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

الكمي في الجسم الحي من التفاعلات بروتين G مستقبلات اقترن به الطيفي ثنائي الفوتون الميكروسكوب

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

بروتوكول لتحديد البروتين البروتين التفاعلات على أساس<sup&gt; 15</sup&gt; N صفها الأيض، مناعي، الكمي والتحوير الطيف الكتلي الانجذاب

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

البروتين البروتين التفاعلات تصور من قبل ثنائي الجزيء الإسفار التكملة في Protoplasts التبغ وأوراق

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

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 ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

التحقيق عالية الكثافة ميكروأرس البروتين وظيفية لكشف البروتين البروتين التفاعلات

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

تطوير مثبطات البروتين البروتين التفاعلات استبدال من خلال: تطبيق لتصميم وتطوير غير ATP مثبطات معلمين تنافسي

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

مناعي لونين الفحص لتحديد نبات الأرابيدوبسيس التفاعلات-DNA البروتين<em&gt; في فيفو</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

دراسة لتفاعلات البروتين البروتين في البحوث أوتوفاجي

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...