نشكر السادس نغوين لقراءة المخطوطات والمساعدة العامة المختبر.

1. التحضير للثقافة الأجرعية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأجرعية سلالة GV3101 حولت سابقا مع متوافق عبارة BiFC ناقلات pEarleyGate201 - YC وpEarleyGate202 YN ، كل منها يحتوي على مزيج من الحكومة الإسرائيلية بناء. </li><li style=";text-align:right;direction:rtl"> تلقيح لمدة 5 مل YEB (5G لحم -1 L استخراج ، 1G الخميرة -1 استخراج L ، L -1 5G ببتون ، 5G السكروز -1 L ، 2mm وMgSO 4 ، ودرجة الحموضة 7.2) ثقافة انصهار بروتين يبني BiFC تحولت مع الأجرعية اختيار المضاد الحيوي المناسب. ثقافة تنمو بين عشية وضحاها في 28 درجة مئوية تهتز في 200 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة 1 مل من ثقافة إلى 1.5 مل العقيمة أنبوب الطرد المركزي. </li><li style=";text-align:right;direction:rtl"> خلايا بيليه في 1000 ز لمدة 10 دقيقة في درجة حرارة الغرفة ، وتجاهل طاف. </li><li style=";text-align:right;direction:rtl"> غسل بيليه بإضافة 1 مل من وسائل الاعلام التسلل (5 ز L -1 مد الجلوكوز ، 50 ملي زارة التربية والعلم (سيغما) ، 2 مم نا 3 ص 4 ، 0.1 ملم acetosyringone) 11. Resuspend بيليه بواسطة pipetting ، وخلايا بيليه مرة أخرى في 1000 لمدة 10 دقيقة ز. </li><li style=";text-align:right;direction:rtl"> كرر الخطوة غسل مرتين أخريين. </li><li style=";text-align:right;direction:rtl"> Resuspend الكرية في وسائل الاعلام تسلل 0.5 مل. </li><li style=";text-align:right;direction:rtl"> قياس الامتصاصية في 600 نانومتر ، وضبط النهائي OD 600-1،0 to 1.2. </li><li style=";text-align:right;direction:rtl"> قسامة مبلغ مساو من تعليق الأجرعية كل بناء جديد في أنبوب مل 1.5 ، دوامة لمدة 10 ثانية. لتسلل واحد ، و 100 ميكرولتر من كل بناء أكثر من كافية. </li><li style=";text-align:right;direction:rtl"> الخليط الأجرعية جاهز الآن للتسلل. </li></ol> 2. تسلل <ol start="11" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وتزرع نباتات N. benthamiana عند درجة 21 ، مع 16 ساعة ضوء و 8 دورات ح الظلام. وينبغي أن تستخدم لخمس محطات لمدة ستة أسابيع من العمر للتسلل. </li><li style=";text-align:right;direction:rtl"> إزالة النباتات من النمو في غرفة ومكان تحت الضوء الأبيض لمدة 1 ساعة قبل السماح infiltratring الثغور لفتح بالكامل. </li><li style=";text-align:right;direction:rtl"> وضع خليط الأجرعية معلق في حقنة 1 مل توبركولين زلة طرف دون الإبرة. </li><li style=";text-align:right;direction:rtl"> مكان غيض من حقنة ضد الجانب السفلي من الأوراق وخفض بلطف المكبس مع دعم مباشر على الجانب العلوي من ورقة مع إصبع واحد. فإن السائل المنتشر من خلال ورقة لأنها تملأ الفراغات الهوائية mesophyllar. </li><li style=";text-align:right;direction:rtl"> تسمية المنطقة تسلل بالقلم علامة لتحديد المستقبل. </li><li style=";text-align:right;direction:rtl"> عندما تسلل مع بنيات مختلفة ، للتأكد من إما تغيير القفازات أو تنظيفها مع الايثانول 70 ٪ بين عمليات التسلل. إذا كان ذلك ممكنا ، وترك مسافة واحدة بين الضلع الأوسط عينات مختلفة لمنع النشطاء من التلوث. </li><li style=";text-align:right;direction:rtl"> مكان النباتات في خزانة النمو في ظل ظروف النمو الطبيعي. </li></ol> 3. رصد إشارة YFP المعاد <ol start="18" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استئصال جزء س 5mm 5mm من نسيج نبات داخل المنطقة تسلل </li><li style=";text-align:right;direction:rtl"> تركيب العينة ، في الماء ، وعلى شريحة ميكروسكوب زجاجية تغطي عينة مع ساترة ودراسة التفاعلات باستخدام المجهر مبائر أو مضان. </li><li style=";text-align:right;direction:rtl"> وينبغي رصد التعبير كل 24 ساعة من وقت تسلل تصل إلى 5 أيام في وقت لاحق فضلا عن أكثر من التعبير / الاتجار يمكن أن ينتج البروتينات الفلورية الانصهار عرض عدة مواقع مختلفة على مسار الزمن. </li></ol> 4. ممثل النتائج : ويرد مثال من البروتين البروتين التفاعل اختباره من قبل مقايسة BiFC في الشكل 1. ويعتقد AtTHP1 وAtSAC3A أن تكون مكونات homolog Arabidopsis مجمع الخميرة TREX - 2. يمكن أن تتفاعل مع AtNUP1 nuclearporin وتسهيل تصدير مرنا 10. وكانت مستنسخة AtTHP1 AtSAC3A في pEarlyGate201 - YC حين تم استنساخ AtNUP1 في YN - pEarlyGate202. تحولت لاحقا الى ثوابت GV3101. وإقران يبني الثلاث للمع 3 التوليفات الممكنة (AtTHP1 + AtNUP1 ؛ AtTHP1 + AtSAC3A ؛ AtNUP1 AtSAC3A +) لعمليات التسلل. ولوحظ في إشارة YFP المعاد تحت 48 ساعة بعد LCSM التسلل. وقد لوحظت اشارات في خلايا البشرة ومحلية في محيط النووية أو البلازما النووية ، وهو ما يتسق مع التعريب شبه الخلوية لهذه البروتينات. لم الضوابط السلبية لا تظهر أي إشارة YFP. <img src="/files/ftp_upload/3473/3473fig1.jpg" alt="الشكل 1" /> الشكل 1. Arabidopsis TREX - 2 مكونات الصادرات مرنا معقدة تتفاعل مع بعضها البعض. ن. تنصهر benthamiana الأوراق ، وشارك في تحويلها مع يبني من الحكومة الإسرائيلية إلى YC - pEarleyGate201 وpEarleyGate202 - YN (كما هو مبين) ، تم تصوير 48-72 ساعة بعد تسلل ، وذلك باستخدام مجهر لايكا TCS confocol SP2. وتظهر الصور كما YFP مبائر المدمجة ومشرق الميدان صور N. البشرة benthamiana خلايا نبات

<ol>
	<li>Ohad, N., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+bimolecular+fluorescence+complementation+(BiFC)+to+assay+protein-protein+interaction+in+plants.">Utilizing bimolecular fluorescence complementation (BiFC) to assay protein-protein interaction in plants.</a> Methods in molecular biology. 655, 347-358 (2010).</li><li>Fang, Y., Spector, D. 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الإعلان عن أي تضارب في المصالح.

BiFC مقايسة هو أداة قوية لدراسة التفاعلات بين البروتينات. خلافا للمقايسة Y2H التقليدية ، ليس فقط BiFC يسمح التصور من البروتين البروتين التفاعلات ، ولكنها توفر أيضا مزيدا من المعلومات من بروتين معقد مثل شبه الخلوية التوطين. أيضا ، فمن الممكن الكشف عن التفاعلات غير مباشرة م...

<table border='1'>
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma</td>
			<td>M2933</td>
		</tr>
		<tr>
			<td>1 mL slip-tip tuberculin syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
	</tbody>
</table>

detection of protein interactions in plant using a gateway compatible bimolecular fluorescence complementation (bifc) system

قمنا بتطوير تقنية BiFC لاختبار التفاعل بين اثنين من البروتينات في الجسم الحي. ويتم تحقيق هذا عن طريق تقسيم البروتين أصفر فلوري (YFP) إلى قسمين شظايا غير متداخلة. هو استنساخ كل جزء في الإطار لجينة من الفائدة. ويمكن بعد هذه التركيبات يشترك في تحويلها إلى benthamiana نيكوتيانا عبر التحول بوساطة الأجرعية ، والسماح للتعبير العبور من البروتينات الانصهار. إعادة تشكيل إشارة YFP يحدث فقط عندما تتفاعل البروتينات التحقيق 1-7. لاختبار والتحقق من تفاعلات البروتين البروتين ، يمكن استخدام BiFC مع اثنين من الخميرة مقايسة (Y2H) المختلطة. وهذا قد كشف عن التفاعلات غير المباشرة التي يمكن التغاضي عنها في Y2H. بوابة التكنولوجيا منصة عالمية تتيح للباحثين المكوك الجينات في المصالح (الحكومة الإسرائيلية) في كتعبير كثيرة وتحليل النظم الفنية و8،9 ممكن. لا يمكن الحفاظ على حد سواء اتجاه القراءة والإطار دون قيد أو باستخدام أنزيمات الربط لجعل التعبير عن استعداد الحيوانات المستنسخة. نتيجة لذلك ، يمكن للمرء أن القضاء على جميع الخطوات إعادة التسلسل لضمان تحقيق نتائج متسقة في جميع أنحاء التجارب. قمنا بإنشاء سلسلة من نواقل عبارة BiFC وY2H التي توفر للباحثين متوافق مع سهلة الاستخدام الأدوات اللازمة لأداء كل من المقايسات وBiFC Y2H 10. هنا ، علينا أن نظهر للتخفيف من استخدام نظامنا BiFC لاختبار البروتين البروتين التفاعلات في N. benthamiana النباتات.

Watch this Scientific Journal Video about Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System at JoVE.com

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

قمنا بتطوير تقنية لاختبار البروتين البروتين التفاعلات في المصنع. يتم تقسيم البروتين أصفر فلوري (YFP) إلى قسمين شظايا غير متداخلة. هو استنساخ كل جزء في الإطار لجينة من الفائدة عن طريق نظام البوابة ، وتمكين التعبير عن البروتينات الانصهار. إعادة تشكيل إشارة YFP يحدث فقط عندما تتفاعل البروتينات التحقيق.

الكشف عن تفاعلات البروتين في النبات باستخدام عبارة الإسفار ثنائي الجزيء المتوافقة التكملة (BiFC) نظام

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

detection-protein-interactions-plant-using-gateway-compatible

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Biology

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

25.2K Views.  University of Western Ontario. قمنا بتطوير تقنية لاختبار البروتين البروتين التفاعلات في المصنع. يتم تقسيم البروتين أصفر فلوري (YFP) إلى قسمين شظايا غير متداخلة. هو استنساخ كل جزء في الإطار لجينة من الفائدة عن طريق نظام البوابة ، وتمكين التعبير عن البروتينات الانصهار. إعادة تشكيل إشارة YFP يحدث ف?...

Video: Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

متجانسة الكشف السريع عن طريق فحوصات البيولوجي المغناطيسي نظام Biosensing التحوير

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

ثنائي الجزيء الإسفار التكملة (BiFC) الفحص عن البروتين البروتين في خلايا البصل التفاعل باستخدام مدفع الجينات هيليوس

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

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

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

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

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

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 ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

الإسفار التكملة ثنائي الجزيء

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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 ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

تدفق تحليل Cytometric من التكملة الإسفار ثنائي الجزيء: عالية الإنتاجية الأسلوب الكمي لدراسة التفاعل البروتين البروتين

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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 ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

تحديد تفاعلات البروتين يجند عن طريق المسح الضوئي التفاضلية Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

تحديد غشاء طوبولوجيا البروتين عن طريق الإسفار البروتياز الحماية (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...