O trabalho em nosso laboratório é suportada por concessões do NIH, National Institute of USDA para Agricultura e Alimentação, NSF, BARD, DOE, e BSF para VC

1. Expressão e de extração das proteínas <ol><li> Diferencialmente tag as proteínas a ser testada para a sua detecção. Rótulo a proteína possa ser imobilizada na membrana (ProIM) com uma tag de um tamanho maior (por exemplo, GST) ea marca não fundido pode ser usado como um controle de imobilizado negativo (ProIMnc). Rótulo a proteína para ser usado como uma sonda solúvel (PROSOL) ou com uma grande ou uma pequena etiqueta. Fusível a mesma marca de uma proteína de controlo negativo adequado solúvel (ProSOLnc) e incluir no ensaio de interação para confirmar a especificidade da ligação detectado. </li><li> Escolher o sistema de expressão de proteína para expressar proteínas marcadas, ou seja, E. coli, baculovírus, etc, dependendo da necessidade de modificações pós-translacionais e os rendimentos potenciais das proteínas. </li><li> Extrato ProIM e ProIM nc do organismo de escolha (passo 1.2) usando SDS-PAGE tampão de carregamento (glicerol 20%, 4% SDS, 20 mM Tris-HCl, pH6.8) contendo coquetel inibidor de protease. Deixe a suspensão de células em temperatura ambiente por 15 min, adicionar 0,5% de 2-mercaptoetanol e ferva por 5 min. </li><li> Extrato PROSOL e PROSOL nc do organismo de escolha (passo 1.2) usando um protocolo padrão 16. Estas proteínas devem ser facilmente solúvel no buffer de ligação (140 mM NaCl, 10 mM, Tris-HCl pH 7,4, 2 mM EDTA, 2 mM DTT, BSA 1%, 0,1% Tween 20) utilizado no passo 4.1. </li><li> Determinar a concentração de proteínas recombinantes utilizando um método padrão, como Bio-Rad kit ensaio de proteína. tais como Bio-Rad kit ensaio de proteína. Se extrato bruto, em vez de proteína purificada, é usado para o ensaio, a estimativa da concentração da proteína de interesse neste extrato, verificando densitometria da banda correspondentes de um SDS-poliacrilamida gel corado com Coomassie Brilliant Blue R-250 e usando conhecida concentrações de BSA como referência. </li></ol> 2. Imobilização de ProIM e ProIMnc na membrana <ol><li> Resolver dois conjuntos de extratos, cada um contendo 1 mg de ProIM e nc ProIM em um gel de poliacrilamida SDS-de acordo com o protocolo padrão 16. </li><li> Após a eletroforese, coloque a transferência do gel para em 100 ml de tampão de transferência (60 mM glicina, 10 mM Tris, 0,0006% SDS, MeOH 20%) e incubar com agitação suave por 20 min em temperatura ambiente. </li><li> Electrotransfer as proteínas contidas no gel para uma membrana de nitrocelulose de acordo com o protocolo padrão 16. </li></ol> 3. Re-folding de proteínas ligadas à membrana <ol><li> Depois electrotransfer, incubar a membrana por 15 min em 15 ml de tampão A (30 mM Tris-HCl pH7.4, 0,05% Tween 20) com agitação suave para remover SDS residual. </li><li> Após a drenagem cuidadosamente, a transferência da membrana para 25 ml de tampão de desnaturação (7 M de cloridrato de guanidina, 2 mM EDTA, 50 mM DTT, 50 mM Tris-HCl pH 8,3). e incubar por 2 horas em temperatura ambiente com agitação suave. (Nota: A membrana de nitrocelulose torna-se opaco quando incubadas no buffer de desnaturação). e incubar por 2 horas em temperatura ambiente com agitação suave. </li><li> Transferência da membrana para 25 ml de TBS (10 mM Tris pH-HCl, 150 mM NaCl, 7,4) e incubar com agitação suave por 5 min (Nota: Durante esta etapa, a membrana recupera a sua cor original branca). </li><li> Transferência da membrana para 25 ml de tampão de ligação (veja o passo 1.2) e incubar a 4 ° C com agitação suave para pernoite. </li></ol> 4. Sondando o ProIM pela PROSOL <ol><li> Corte da membrana em duas faixas, ambas contendo ProIM e ProIM nc. </li><li> Faça as soluções de hibridização PROSOL e ProSOLnc diluindo a preparação com 10-10 mg de qualquer PROSOL ou ProSOLnc em 10 ml de tampão de ligação nova. Transferência de cada membrana na solução de hibridização PROSOL ou ProSOLnc e incubar por 1,5 h à temperatura ambiente com agitação suave. </li><li> Remova as membranas das soluções de hibridização e enxaguar três vezes por 15 min cada um em TBS. </li></ol> 5. Visualizar a interação proteína-proteína por immunoblotting <ol><li> Bloco da membrana, com 2,5% de leite desnatado em TBST (10 mM Tris-HCl, 140 mM NaCl, 0,05% Tween 20, pH 7,4) por 1 h em temperatura ambiente. Diluir o anticorpo primário (anti-anticorpo de coelho strepII policlonal) em 0,5% de leite desnatado em TBST na concentração recomendada pelo fabricante. </li><li> Coloque as membranas bloqueados na solução de anticorpos, e incubados por 1 h em temperatura ambiente ou para a noite a 4 ° C com agitação suave. </li><li> Enxaguar as membranas em 20 TBST ml por 15 min, e duas vezes por 5 min em temperatura ambiente com agitação suave. </li><li> Diluir o anticorpo secundário (anti-IgG de coelho) conjugado com peroxidase de rábano (HRP) em 0,5% de leite desnatado em TBST na concentração recomendada pelo fabricante. Coloque o membranas no anticorpo secundáriosolução e incubar por 1 h em temperatura ambiente com agitação suave. </li><li> Enxaguar as membranas em 20 TBST ml por 15 min, e duas vezes por 5 min em temperatura ambiente com agitação suave. Após o enxágüe final em TBS, visualizar a interação proteína-proteína utilizando um substrato quimioluminescência HRP (por exemplo, Millipore Immobilon ocidental substrato quimioluminescente HRP). </li><li> Sonda as membranas mesmo com o anticorpo primário para a marca fundida a ProIM, seguido pelo anticorpo secundário apropriado conforme descrito nas etapas 5,1-5,4. Visualize ProIM e ProIMnc como descrito no passo 5,5 para validar a identidade das bandas obtidas no ensaio de sobreposição de proteína de membrana (passo 5.5). Na maioria dos casos, stripping não é necessário, porque o sinal obtido por este passo é muito mais forte do sinal residual obtido a partir do passo 5.5. </li></ol> 6. Resultados representativos: A interação ANK-MP foi observado por BiFC em células epidérmicas de tabaco (Figura 1A). MP porque é uma proteína altamente insolúvel quando expressos em bactérias ou em plantas, a proteína de membrana ensaio overlay foi adotado para validar essa interação in vitro (Figura 1B). Extratos de proteínas contendo 1 mg de GST-MP (ProIM) ou GST não fundido (ProIM nc) foram resolvidas por SDS-eletroforese em gel de poliacrilamida, seguida por electrotransfer a uma membrana de nitrocelulose. Quando estes ProIMs foram sondados com solúveis ANK-strepII (PROSOL), GST-MP, mas não GST não fundido, exibiu ligação (Figura 1B, faixas 1, 2, comparar com pistas 5, 6). Além disso, quando o mesmo conjunto de ProIMs foram sondados com uma ProSOLnc independentes, ou seja, quinase NADH citoplasmático Arabidopsis tag strepII (NADH3-strepII), nenhuma ligação não foi observada, demonstrando a especificidade da interação ANK-MP (Figura 1B, pistas 3, 4). <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="Figura 1"/> Figura 1. Ligação específica do tabaco ANK para TMV MP in vivo e in vitro. (A) interação ANK-MP em células vivas do tabaco epidérmica como detectado pelo BiFC. YFP sinal forte foi reconstruído quando MP e ANK, fundido ao C-terminal e N-terminal metades de YFP, respectivamente, foram co-expressos na epiderme do tabaco seguintes microbombardment de genes a sua codificação. Este sinal BiFC acumulado em puncta na periferia das células, que são de diagnóstico de plasmodesmos 15. (B) interação ANK-MP in vitro como detectado pelo teste de proteína de membrana overlay. Extratos de proteínas contendo 1 mg de GST-MP (ProIM) ou GST não fundido (ProIMnc) foram resolvidas em um 15% gel de poliacrilamida SDS, seguido por electrotransfer em uma membrana de nitrocelulose. A GST-MPProIM e GSTProIMnc foram incubadas com 0,5 mg / ml de ANK-strepII (PROSOL), e ANK ligação foi detectado pela sondagem da membrana com anticorpo anti-coelho strepII policlonal, seguido de anticorpos IgG anti-coelho M + secundário conjugado com HRP (faixas 1 e 2). Nem GST-MPProIM nem GSTProIMnc interagiu com uma proteína não relacionada, Arabidopsis quinase NADH citoplasmático, fundida ao tag strepII (ProSOLnc, faixas 3 e 4). A identidade da banda observada neste ensaio foi confirmada por sondagem a membrana com anticorpos anti-GST (faixas 5 e 6). Quando a membrana foi tratado com tampão de desnaturação sem ser lavada com tampão A, a ligação do GST-MP para ANK é perdido, enquanto que as proteínas não identificadas contidas no GST-MP e extratos de proteína GST contining reagiu com ANK-strepII, demonstrando a importância da etapa de 3,1 antes do processo de desnaturação (faixas 7 e 8).

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Esta abordagem é adequada para testar interações proteína-proteína entre as combinações das proteínas, quando pelo menos um dos quais as proteínas é facilmente solúvel no buffer de ligação, e foi aplicado com sucesso em outra combinação de proteínas 17,18. O iInteractions entre as proteínas que são insolúveis nestas condições não pode ser testada por este protocolo. Além disso, redobrando bem-sucedida de ProIM é fundamental para o ensaio. Lavar a membrana e...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Kit ensaio de proteína </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Proteinase cocktail inibidor </td><td> SIGMA </td><td> S8820 </td></tr><tr><td> Mini-PROTEAN sistema </td><td> Bio-RAD </td><td> 165-8000 </td></tr><tr><td> Semi-seco western blotting SD sistema electrotransfer </td><td> Bio-RAD </td><td> 170-3940 </td></tr><tr><td> Anti-IgG de coelho conjugado com peroxidase de rabanete de cavalo </td><td> GenScript </td><td> A00098 </td></tr><tr><td> Anti-GST anticorpo policlonal de coelho </td><td> GenScript </td><td> A00097 </td></tr><tr><td> Anti-strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> BioTrace, NT membrana de transferência de nitrocelulose </td><td> Pall Scientific </td><td> 27377-000 </td></tr><tr><td> Immobilon ocidental substrato quimioluminescente HRP </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

Interações entre proteínas diferentes validar é vital para a investigação de suas funções biológicas em nível molecular. Existem vários métodos, tanto in vitro como in vivo, para avaliar a ligação às proteínas, e pelo menos dois métodos que complementam as deficiências do outro deve ser conduzido para obter conclusões confiáveis. Para um ensaio in vivo, a complementação bimolecular de fluorescência ensaio (BiFC) representa a abordagem mais popular e menos invasivo que permite detectar a interação proteína-proteína dentro das células vivas, bem como identificar a localização intracelular de proteínas que interagem a 1,2. Neste ensaio, as metades não fluorescente N-e C-terminal da GFP, ou suas variantes são fundidos às proteínas testadas, e quando as duas proteínas de fusão são reunidos devido às interações das proteínas testadas &quot;, o sinal fluorescente é reconstituído 06/03 . Porque seu sinal é facilmente detectável por microscopia de epifluorescência ou confocal, BiFC emergiu como uma poderosa ferramenta de escolha entre os biólogos para estudar sobre interações proteína-proteína em células vivas 3. Este ensaio, entretanto, às vezes pode produzir resultados falsos positivos. Por exemplo, o sinal fluorescente pode ser reconstituído por dois fragmentos de GFP dispostos, tanto quanto 7 nm um do outro devido a fechar embalagem, em um pequeno compartimento subcelular, e que, devido a interações específicas 7. Devido a essas limitações, os resultados obtidos a partir de tecnologias de imagem ao vivo de células deve ser confirmado por uma abordagem independente, baseada em um princípio diferente para a detecção de interações proteína. Co-imunoprecipitação (IP-Co) ou glutationa transferase (GST) suspenso ensaios representam tais métodos alternativos que são comumente usados ​​para analisar interações proteína-proteína in vitro. No entanto, estes ensaios iin, no entanto, as proteínas testado deve ser facilmente solúvel no buffer que supportsused para a reação de ligação. Portanto, interações específicas, envolvendo uma proteína insolúvel não podem ser avaliados por estas técnicas. Aqui, nós ilustrar o protocolo para o ensaio overlay proteína de membrana de ligação, que contorna esta dificuldade. Nesta técnica, a interação entre as proteínas solúveis e insolúveis podem ser seguramente testado, porque uma das proteínas é imobilizado em uma matriz de membrana. Este método, em combinação com experimentos in vivo, tais como BiFC, fornece uma abordagem confiável para investigar e caracterizar as interações entre proteínas fielmente solúveis e insolúveis. Neste artigo, a ligação entre o vírus do mosaico do tabaco (TMV) proteína de movimento (MP), que exerce várias funções durante o transporte célula a célula-viral 8-14, e um interator planta recentemente identificado celulares, tabaco anquirina repetir proteína contendo (ANK ) 15, é demonstrado usando esta técnica.

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

Interação proteína-proteína testes é indispensável para dissecção de funcionalidade da proteína. Aqui, nós apresentamos uma<em&gt; In vitro</em&gt; Ensaio de proteína-proteína de ligação à sonda uma membrana de proteína-imobilizada com uma proteína solúvel. Este ensaio fornece um método confiável para testar a interação entre uma proteína insolúvel e uma proteína em solução.

Protein Assay Overlay membrana: um protocolo para testar Interação entre proteínas solúveis e insolúveis 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

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Biology

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

22.0K visualizações.  State University of New York. Interação proteína-proteína testes é indispensável para dissecção de funcionalidade da proteína. Aqui, nós apresentamos uma In vitro Ensaio de proteína-proteína de ligação à sonda uma membrana de proteína-imobilizada com uma proteína solúvel. Este ensaio fornece um método confiável para testar a interação entre uma proteína insolúvel e uma proteína em solução.

Vídeo: 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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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

Biologia

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)

Bimolecular de fluorescência de Complementação Assay (BiFC) para Interação Proteína-Proteína em células de cebola Utilizando o Gene Gun 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.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

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.

A utilização de um robô para alta capacidade de cristalização de proteínas de membrana em mesofases lipídicas

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.

A metilação in vitro Ensaio de Proteína Estudo Arginina metilação

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).

Genome-wide proteína-proteína Interaction Triagem por proteína-fragmento de Complementação Assay (PCA) em células vivas

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.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<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.

Luciferase complementação Imaging ensaio em folhas de Nicotiana benthamiana de transitoriamente determinação dinâmica da interação da proteína-proteína

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.

Determinação da interação tripartite entre dois monômeros de um fator de transcrição da caixa MADS e uma proteína de sensor de cálcio por Ensaio 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 ...

Produção e imagem de vesículas cheias de proteínas recombinantes de 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.

Autor em destaque: A produção de proteínas 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.