Le travail de notre laboratoire est soutenu par des subventions du NIH, Institut national de l&#39;USDA pour l&#39;alimentation et l&#39;agriculture, de la NSF, BARD, le DOE, et BSF au VC

1. Expression et l&#39;extraction des protéines <ol><li> Différentiellement tag les protéines d&#39;être testés pour leur détection. Étiquette à la protéine d&#39;être immobilisée sur la membrane (ProIM) avec une étiquette de plus grande taille (par exemple, TPS), et la balise sans fusible peut être utilisé comme un contrôle négatif immobilisé (ProIMnc). Étiquette à la protéine d&#39;être utilisé comme une sonde solubles (PROSOL) avec soit une grande ou une petite étiquette. Fusible la même étiquette à une protéine de contrôle négatif appropriés solubles (ProSOLnc) et inclure dans le test d&#39;interaction pour confirmer la spécificité de la liaison détectée. </li><li> Choisissez le système d&#39;expression de protéines pour exprimer des protéines marquées, à savoir, E. coli, baculovirus, etc, selon l&#39;exigence de modifications post traductionnelles et les rendements potentiels des protéines. </li><li> Extrait ProIM et ProIM nc de l&#39;organisme de son choix (étape 1.2) en utilisant un tampon de charge SDS-PAGE (glycérol 20%, 4% SDS, 20 mM Tris-HCI, pH 6,8) contenant un cocktail d&#39;inhibiteur de protéinase. Laisser la suspension cellulaire à température ambiante pendant 15 min, ajouter 0,5% de 2-mercaptoéthanol et faire bouillir pendant 5 min. </li><li> Extrait PROSOL et PROSOL nc de l&#39;organisme de son choix (étape 1.2) en utilisant un des protocoles standard de 16. Ces protéines doivent être facilement solubles dans le tampon de liaison (140 mM NaCl, 10 mM, Tris-HCl pH 7,4, EDTA 2 mM, 2 mM DTT, 1% de BSA, 0,1% Tween 20) utilisé dans l&#39;étape 4.1. </li><li> Déterminer la concentration de protéines recombinantes utilisant une méthode standard, tels que Bio-Rad kit de dosage de protéines. tels que Bio-Rad kit de dosage de protéines. Si l&#39;extrait brut, plutôt que la protéine purifiée, est utilisée pour le dosage, l&#39;estimation de la concentration de la protéine d&#39;intérêt dans cet extrait en scannant la densitométrie de la bande correspondante sur une SDS-polyacrylamide gel coloré de Coomassie Brilliant Blue R-250 et en utilisant connus Les concentrations de BSA comme référence. </li></ol> 2. Immobilisation de ProIM et ProIMnc sur la membrane <ol><li> Résoudre les deux ensembles d&#39;extraits, chacun contenant 1 ug d&#39;ProIM et ProIM nc sur un gel SDS-polyacrylamide selon le protocole standard 16. </li><li> Après l&#39;électrophorèse, placer le transfert du gel dans 100 ml de tampon de transfert (60 mM de glycine, 10 mM Tris, 0,0006% de SDS, 20% MeOH) et incuber en agitant doucement pendant 20 min à température ambiante. </li><li> Électrotransfert des protéines contenues dans le gel d&#39;une membrane de nitrocellulose selon le protocole standard 16. </li></ol> 3. Re-repliement des protéines membranaires <ol><li> Après électrotransfert, incuber la membrane pendant 15 min dans 15 ml de tampon A (30 mM Tris-HCl pH 7,4, 0,05% Tween 20) avec une agitation douce pour éliminer les résidus de SDS. </li><li> Après la vidange attentivement, le transfert de la membrane à 25 ml de tampon de dénaturation (7 M chlorhydrate de guanidine, 2 mM EDTA, 50 mM DTT, 50 mM Tris-HCl pH 8,3). et incuber pendant 2 heures à température ambiante avec agitation douce. (Remarque: La membrane de nitrocellulose devient opaque lorsqu&#39;elle est incubée dans le tampon de dénaturation). et incuber pendant 2 heures à température ambiante avec agitation douce. </li><li> Transférer la membrane à 25 ml de TBS (10 mM Tris-HCl, NaCl 150 mM, pH 7,4) et incuber en agitant doucement pendant 5 min (Note: Durant cette étape, la membrane reprend sa couleur blanche originale). </li><li> Transférer la membrane à 25 ml de tampon de liaison (voir étape 1.2) et incuber à 4 ° C avec agitation douce pendant la nuit. </li></ol> 4. Sonder les ProIM par PROSOL <ol><li> Couper la membrane en deux bandes, à la fois contenant et ProIM ProIM nc. </li><li> Faire les solutions d&#39;hybridation et de PROSOL ProSOLnc par dilution de la préparation avec mg 1-10 de PROSOL soit ou ProSOLnc dans 10 ml de tampon de liaison douce. Transfert chaque membrane dans la solution d&#39;hybridation ou de PROSOL ProSOLnc et incuber pendant 1,5 h à température ambiante avec agitation douce. </li><li> Retirer les membranes des solutions d&#39;hybridation et rincer trois fois pendant 15 minutes dans chacune des SCT. </li></ol> 5. Visualisation interaction protéine-protéine par immunoblot <ol><li> Bloc de la membrane avec du lait écrémé 2,5% dans TBST (10 mM Tris-HCl, 140 mM de NaCl, 0,05% Tween 20, pH 7,4) pendant 1 h à température ambiante. Diluer l&#39;anticorps primaire (anti-strepII anticorps polyclonal de lapin) dans le lait écrémé 0,5% dans TBST à la concentration recommandée par le fabricant. </li><li> Placer les membranes bloquées dans la solution d&#39;anticorps, et incubées pendant 1 h à température ambiante ou pour une nuit à 4 ° C avec agitation douce. </li><li> Rincer les membranes dans 20 ml TBST pendant 15 min, et deux fois pendant 5 min à température ambiante avec agitation douce. </li><li> Diluer l&#39;anticorps secondaire (anti-IgG de lapin) conjugué à la peroxydase de raifort (HRP) dans le lait écrémé 0,5% dans TBST à la concentration recommandée par le fabricant. Placer les membranes dans l&#39;anticorps secondairesolution et incuber pendant 1 h à température ambiante avec agitation douce. </li><li> Rincer les membranes dans 20 ml TBST pendant 15 min, et deux fois pendant 5 min à température ambiante avec agitation douce. Après le rinçage final dans le TBS, de visualiser l&#39;interaction protéine-protéine en utilisant un substrat HRP chimiluminescence (par exemple, Millipore Immobilon ouest chimioluminescence HRP substrat). </li><li> Sondez les mêmes membranes avec l&#39;anticorps primaire pour l&#39;étiquette fusionnée à ProIM, suivie par l&#39;anticorps secondaire approprié tel que décrit dans les étapes 5.1 à 5.4. Visualisez ProIM et ProIMnc comme décrit dans l&#39;étape 5.5 de valider l&#39;identité des bandes obtenues dans le dosage de superposition des protéines membranaires (étape 5.5). Dans la plupart des cas, le décapage n&#39;est pas nécessaire, parce que le signal obtenu par cette étape est beaucoup plus forte que signal résiduel obtenu à l&#39;étape 5.5. </li></ol> 6. Les résultats représentatifs: L&#39;interaction ANK-MP a été observée par BiFC dans les cellules de tabac épidermique (figure 1A). Parce que MP est une protéine hautement insoluble lorsqu&#39;il est exprimé dans des bactéries ou des plantes, le dosage de superposition des protéines membranaires a été adoptée afin de valider cette interaction in vitro (figure 1B). Des extraits protéiques contenant 1 ug de GST-MP (ProIM) ou non condensés TPS (ProIM NC) ont été résolus par SDS-polyacrylamide gel d&#39;électrophorèse, suivie par électrotransfert sur ​​membrane de nitrocellulose. Lorsque ces ProIMs ont été sondés avec solubles ANK-strepII (PROSOL), TPS-MP, mais pas non fusionnée TPS, exposée liaison (figure 1B, les pistes 1, 2, comparer aux lignes 5, 6). En outre, lorsque le même ensemble de ProIMs ont été sondés avec une ProSOLnc indépendantes, c&#39;est à dire, Arabidopsis kinase cytoplasmique NADH taggés strepII (NADH3-strepII), aucune liaison n&#39;a été observée, démontrant la spécificité de l&#39;interaction ANK-MP (figure 1B, les voies 3, 4). <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="Figure 1"/> Figure 1. Liaison spécifique de tabac ANK au TMV MP in vivo et in vitro. (A) ANK-MP interaction dans des cellules vivantes du tabac épidermique détectée par BiFC. Forte de signal YFP a été reconstruit en MP et ANK, fusionnée à C-terminale et N-terminale moitiés de la YFP, respectivement, ont été co-exprimés dans l&#39;épiderme du tabac suivants microbombardment de leurs gènes codant. Ce signal BiFC accumulés dans lacrymaux, à la périphérie des cellules, qui sont de diagnostic des plasmodesmes 15. (B) ANK-MP interaction in vitro, tel que détecté par le dosage des protéines membranaires de superposition. Des extraits protéiques contenant 1 ug de GST-MP (ProIM) ou non condensés TPS (ProIMnc) ont été résolus sur un 15% gel SDS-polyacrylamide, suivie par électrotransfert sur membrane de nitrocellulose. La TPS et la MPProIM GSTProIMnc ont été incubées avec 0,5 pg / ml de ANK-strepII (PROSOL), et ANK contraignante a été détectée en sondant la membrane avec l&#39;anticorps de lapin anti-strepII polyclonaux, suivis par les anti-IgG de lapin + M anticorps secondaire conjugué à la HRP (voies 1 et 2). Ni la TPS ni MPProIM GSTProIMnc interagi avec une protéine non liée, chez Arabidopsis kinase cytoplasmique NADH, fusionnée à la balise strepII (ProSOLnc, pistes 3 ​​et 4). L&#39;identité de la bande observée dans cet essai a été confirmée en sondant la membrane avec anticorps anti-GST (pistes 5 et 6). Lorsque la membrane a été traité avec tampon de dénaturation, sans être lavés avec le tampon A, la liaison de la TPS-MP au ANK est perdu, tandis que les protéines non identifiées contenues dans le TPS-MP et des extraits de protéines contenant leurs TPS réagi avec ANK-strepII, démontrant l&#39;importance de l&#39;étape 3.1, avant le processus de dénaturation (voies 7 et 8).

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Aucun conflit d&#39;intérêt déclaré.

Cette approche est appropriée pour les tests interactions protéine-protéine entre les combinaisons des protéines, quand au moins un dont les protéines est facilement soluble dans le tampon de liaison, et a été appliquée avec succès à une autre combinaison de protéines 17,18. Le iInteractions entre les protéines qui sont insolubles dans ces deux conditions ne peuvent pas être testés par ce protocole. Aussi, le repliement de la réussite ProIM est critique pour le dosa...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Le numéro de catalogue </td></tr><tr><td> Kit de dosage de protéines </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Cocktail inhibiteur de protéinase </td><td> SIGMA </td><td> S8820 </td></tr><tr><td> Mini-Protean système de </td><td> Bio-Rad </td><td> 165-8000 </td></tr><tr><td> Demi-sec western blot SD électrotransfert système de </td><td> Bio-Rad </td><td> 170-3940 </td></tr><tr><td> Anti-IgG de lapin conjugué à la peroxydase de raifort </td><td> GenScript </td><td> A00098 </td></tr><tr><td> Anti-TPS de lapin d&#39;anticorps polyclonaux </td><td> GenScript </td><td> A00097 </td></tr><tr><td> Anti-strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> BioTrace, la membrane de nitrocellulose NT transfert </td><td> Pall scientifique </td><td> 27377-000 </td></tr><tr><td> Immobilon ouest chimioluminescence HRP substrat </td><td> Millipore </td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

Validation des interactions entre différentes protéines est essentielle pour l&#39;étude de leurs fonctions biologiques au niveau moléculaire. Il existe plusieurs méthodes, à la fois in vitro et in vivo, pour évaluer la liaison aux protéines, et au moins deux méthodes qui se complètent les lacunes de l&#39;autre devrait être menée afin d&#39;obtenir des données fiables. Pour un essai in vivo, la complémentation bimoléculaires de fluorescence (BiFC) test représente l&#39;approche la plus populaire et la moins invasive qui permet de détecter les interactions protéine-protéine dans les cellules vivantes, ainsi que d&#39;identifier la localisation intracellulaire de l&#39;interaction des protéines 1,2. Dans cet essai, non fluorescent moitiés N-et C-terminale de la GFP ou ses variantes sont fusionnés aux protéines testées, et quand les deux protéines de fusion sont réunies en raison des interactions des protéines testées », le signal fluorescent est reconstitué 3-6 . Parce que son signal est facilement détectable par microscopie à épifluorescence ou confocale, BiFC a émergé comme un puissant outil de choix parmi les biologistes cellulaires pour étudier à propos interactions protéine-protéine dans 3 cellules vivantes. Ce test, cependant, peuvent parfois produire des résultats faux positifs. Par exemple, le signal fluorescent peut être reconstituée par deux fragments GFP réglée autant que 7 nm de l&#39;autre en raison de fermer l&#39;emballage dans un petit compartiment subcellulaire, plutôt qu&#39;en raison d&#39;interactions spécifiques 7. En raison de ces limitations, les résultats obtenus à partir des technologies d&#39;imagerie cellulaire direct doit être confirmé par une approche indépendante basée sur un principe différent pour détecter les interactions entre protéines. Co-immunoprécipitation (Co-IP) ou le glutathion transférase (GST) déroulant tests représentent ces méthodes alternatives qui sont couramment utilisées pour analyser les interactions protéine-protéine in vitro. Toutefois, dDans ces essais, cependant, les protéines doivent être testés facilement soluble dans le tampon qui supportsused pour la réaction de liaison. Par conséquent, des interactions spécifiques impliquant une protéine insoluble ne peut être évaluée par ces techniques. Ici, nous illustrons le protocole pour l&#39;essai de superposition protéine membranaire contraignant, qui contourne cette difficulté. Dans cette technique, l&#39;interaction entre les protéines solubles et insolubles peuvent être testés de manière fiable, car l&#39;une des protéines est immobilisé sur une matrice de la membrane. Cette méthode, en combinaison avec les expériences in vivo, comme BiFC, offre une approche fiable pour étudier et caractériser les interactions entre les protéines fidèlement solubles et insolubles. Dans cet article, liant entre le virus de la mosaïque du tabac (TMV) protéine de mouvement (MP), qui exerce de multiples fonctions au cours virale de cellule à cellule de transport 8-14, et un interacteur usine récemment identifié cellulaires, tabac ankyrine répétées contenant des protéines (ANK ) 15, est démontrée en utilisant cette technique.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Test interaction protéine-protéine est indispensable pour la dissection de la fonctionnalité des protéines. Ici, nous introduisons une<em&gt; In vitro</em&gt; Protéine-protéine test de liaison de sonder une membrane immobilisée protéine avec une protéine soluble. Ce test fournit une méthode fiable pour tester l&#39;interaction entre une protéine insoluble et une protéine en solution.

Essai de superposition des protéines membranaires: Un protocole pour tester l&#39;interaction entre les protéines solubles et insolubles In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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Research

JoVE Journal

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

22.0K vues.  State University of New York. Test interaction protéine-protéine est indispensable pour la dissection de la fonctionnalité des protéines. Ici, nous introduisons une In vitro Protéine-protéine test de liaison de sonder une membrane immobilisée protéine avec une protéine soluble. Ce test fournit une méthode fiable pour tester l'interaction entre une protéine insoluble et une protéine en solution.

Vidéo: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

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

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

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

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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

Recherche

Biologie

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)

Bimoléculaire complémentation de fluorescence (BiFC) Dosage pour les interactions protéine-protéine dans les cellules d&#39;oignon Utilisation du pistolet à gènes Helios

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

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un essai en phase liquide d&#39;affinité de capture utilisant des billes magnétiques pour étudier interaction protéine-protéine: Le poliovirus Exemple-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Utilisation d&#39;un robot pour haut-débit cristallisation des protéines membranaires dans des mésophases lipidiques

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro test de Méthylation à étudier des protéines méthylation de l&#39;arginine

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

L&#39;échelle du génome interaction protéine-protéine Le dépistage par Protein-fragment complémentation Assay (PCA) dans les cellules vivantes

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein base Expression dépistage des protéines membranaires dans<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Analyse de l’imagerie de la complémentation de luciférase dans les feuilles de Nicotiana benthamiana pour transitoirement déterminant la dynamique Interaction protéine-protéine

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

Détermination de l’interaction tripartite entre deux monomères d’un facteur de transcription MADS-box et d’une protéine de capteur de calcium par essai BiFC-FRET-FLIM

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

Production et imagerie de vésicules remplies de protéines recombinantes à partir d’E. coli

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Pleins feux sur l’auteur : La production de protéines recombinantes 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.