Agradecemos a drs. Steve Monte e Zhang Xiaoning para o 35S:: GFP pGlowbug construir e Doutorado da Irmandade Hokensen para CH Pesquisa em laboratório Z. L&#39;s é apoiado pela National Science Foundation EUA (IOB0616096 e MCB0744752). ZL é parcialmente financiado pela Universidade de Maryland Estação Experimental Agrícola.

I. preparação Plasmid O DNA plasmídeo usado para bombardeio deve ser de alta pureza e em uma concentração de 500ng/μl ou superior. Para BiFC envolvendo duas construções plasmídeo, 25 mcg de cada plasmídeo é necessário. Em outras palavras, 1:1 molar dos dois plasmídeos devem ser misturadas para dar origem a 50 mg DNA total plasmídeo para a preparação de cartuchos. Esteja consciente de que a adição de mais DNA do que 50 mg pode causar aglomeração das partículas de ouro e deve ser evitado. Comercialmente disponíveis kits de extração de DNA plasmídeo são recomendados para isolar DNA plasmídeo. Vetores plasmídeo deve ser relativamente pequeno em tamanho, como pUC19. Nós SEU clonado e LUH cDNA em pUC19-SPYNE e pUC19-Spyce, respectivamente 3. Vetores binários para Agrobacterium mediada transformação são grandes demais e inadequado para o bombardeio. II. Cartucho de preparação Preparação cartucho envolve as partículas de ouro com revestimento de DNA plasmidial e carregá-los em tubos de plástico que é posteriormente cortada em pedaços meia polegada de comprimento, que pode ser carregado no gene titulares cartucho de arma. Preparação cartucho é descrito em grandes detalhes em um artigo separado Jove 4 e é, portanto, não descritos aqui. Para bombardeio de células da cebola, recomendamos mistura de 50 mg de DNA plasmidial com 12,5 mg de partículas de ouro por preparação, que rende cerca de até 50 cartuchos de 1 mg DNA/0.25 mg de ouro / cartucho. Cada cartucho é de um tiro. As partículas de ouro pode ser 0,6 mm ou 1,0 m de diâmetro (Cat # 1652262 ou Cat # 1652263; Biorad). Também recomendamos o uso de 0,5 mg / ml solução PVP para a preparação do cartucho. Para BiFC, combine os dois DNAs plasmídeo de cada parceiro interagindo putativo na preparação mesmo cartucho. Após a preparação do cartucho, é importante para testar os cartuchos, disparando os cartuchos recém-feitos a uma pressão de hélio entre 150 a 200 psi em um Parafilm quadrado que é enrolado em torno de uma placa de Petri. Isso irá testar se as partículas de ouro podem ser movidos de forma eficiente dos cartuchos, você deve ver partículas de ouro desmaiar na Parafilm. Isso lhe dá uma idéia da área que cada tiro irá abranger. Você pode observar diferentes quantidades de partículas de ouro impelido para o Parafilm em diferentes pressões de hélio (a faixa 150-200 psi). Se não há diferença significativa, escolha a menor pressão de bombardeio. III. Preparar os tecidos de cebola No dia da gravação, retire a pele externa de uma cebola grande amarelo. Usando uma lâmina limpa e afiada, corte 4-5 camadas profundas uma área retangular de cerca de 2 X 8 cm. Retire o tecido retangular cebola, descarte o exterior duas camadas, corte as camadas restantes de cebola em pedaços sobre 2x1.5 cm. Coloque os pedaços de cebola em um prato de Petri contendo papel de filtro (Whatman 3MM de papel cortado em círculos) umedecido com água estéril. Cubra o prato de Petri e eles estão prontos para ir. IV. Bombardeamento <ol><li> Carregar os cartuchos para o branco titulares cartucho redondo. Cartuchos contendo diferentes construções plasmídeo deve ser carregado em titulares cartucho diferente. Cada suporte do cartucho pode armazenar até 12 cartuchos por 12 tiros. </li><li> Insira a bateria e o forro barril fresco no Gun Gene Helios. Certifique-se que tanto o forro do cano da arma e tem respectivos o-rings em anexo. </li><li> Primeiro, insira um cartucho vazio titular na arma gene, com a marca # 12 no suporte do cartucho de frente para o topo da arma. Suporte do cartucho avançar uma ou duas vezes para ter certeza que está inserido corretamente. </li><li> Anexar arma para o tanque de hélio e ajustar a pressão entre 150 a 200 psi. </li><li> Usa protetor auricular e arma de fogo algumas vezes com o suporte do cartucho vazio para limpar a câmara da arma e para se certificar de que a pressão está estável. </li><li> Remova o suporte do cartucho vazio e coloque um suporte do cartucho carregado. Avançar o suporte do cartucho para que o cartucho que você deseja para o fogo está posicionado na parte inferior (06:00) do suporte do cartucho. Fotografar um pedaço de cebola aqui fornece uma forma de controle negativa para fluorescência de fundo. </li><li> Coloque os pedaços de cebola no meio de um prato vazio Petri com a camada mais interna da cebola voltada para cima. Resto do forro a arma gene barril na placa de Petri e disparar a arma pressionando o botão lateral, puxando o gatilho. Transfira a cebola bombardeados com uma placa de Petri contendo papel filtro úmido 3MM. </li><li> Avançar para o próximo cartucho e fogo um pedaço de cebola diferente. Repita este passo até 4-5 pedaços de cebola são filmadas. Cada pedaço de cebola é baleado uma vez. </li><li> Mudar para um suporte do cartucho carregado com diferentes cartuchos contendo uma combinação plasmídeo ou diferentes diferentes plasmídeo. Lembre-se de mudar o forro barrils bem para evitar a contaminação. Atire 4-5 pedaços de cebola. </li><li> Após bombardeio, retornar todas as cebolas aos pratos pertinência. Cobrir as placas de Petri com tampa. Envolvê-los com Parafilm para evitar a secagem. Incubar os pedaços de cebola em temperatura ambiente no escuro por 16-48 horas. </li><li> Desligue a pressão do gás e liberação antes de separar a arma gene do tanque de hélio. Liner desapertar o barril, e remover o suporte do cartucho e os cartuchos usados ​​(ou não). </li><li> Limpeza titulares cartucho e forros barril, submergindo-os num copo com água e sabão. Coloque o copo num banho e sonicador sonicate por 20 minutos. Depois, lave-os com água para remover todos os resíduos de sabão. Mergulhe-os em etanol 70% por uma hora e depois seque-os sobre papel absorvente. </li></ol> V. Observação <ol><li> Após incubação pedaços de cebola bombardeados durante 16-20 horas à temperatura ambiente, podemos observar GFP ou YFP fluorescência usando um microscópio de fluorescência, como a Zeiss Axio Observer.z1 utilizados neste estudo. </li><li> Use uma pinça com extremidades planas lentamente a casca de uma única camada celular fora da epiderme interna da cebola (a camada que se confronta diretamente com a arma durante o bombardeio). Coloque as cascas em uma gota de água em um slide. Adicionar lamela mas tenha cuidado para minimizar bolhas. </li><li> Em campo claro do microscópio, verifique primeiro para streaming citoplasmática para garantir que as células estão vivas. Para isso, expor o slide para a luz brilhante campo por alguns minutos para aquecer o tecido de cebola e depois olhar para plastídios em movimento. Streaming de citoplasmática nem sempre é fácil de ver, por isso não desanime se você não pode ver nenhuma. </li><li> Para a detecção de fluorescência, use a excitação apropriada e filtros de detecção de GFP ou YFP. É importante incluir controles negativos, tais como pedaços de cebola tiro com um plasmídeo não-fluorescente. Sinais fracos amarela pode aparecer a partir de células feridos, bolhas de ar, ou detritos. Também é importante incluir controlos positivos. Um plasmídeo contendo repórter constitutivamente expressa fluorescentes, como 35S:: GFP é um excelente controle positivo. Sinais visíveis a partir de células GFP cebola epidérmica (Figura 1A, D) indicou que os cartuchos, os tecidos, cebola e os disparos de armas gene está devidamente preparado e executado. Posteriormente, observou-se as interações entre SEU-pSPYNE e LUH-pSPYCE em pedaços de cebola bombardeados com cartuchos contendo uma mistura de 35S:: SEU-pSPYNE e 35S:: LUH-pSPYCE DNAs. Fluorescência YFP fraca foi observada no núcleo só (Figura 1 B, E), indicando a interação física direta entre SEU LUH e que trouxe os fragmentos YFP dividir juntos no núcleo. Note que o sinal da YFP BiFC é significativamente mais fraca do que o sinal da GFP 35S:: GFP. </li></ol> Resultados representante Em nosso estudo aqui relatado, o 35S:: GFP controle positivo plasmídeos emite fluorescência forte que preenche toda a célula, incluindo tanto o núcleo eo citoplasma (Figura 1A, D). Proteína GFP é pequeno o suficiente para difundir para o núcleo, sem um sinal de localização nuclear. Em contraste, SEU e LUH são dois fatores de transcrição de Arabidopsis mostrado anteriormente para interagir em uma levedura híbrida de dois ensaio 2. SEU-pSPYNE (SEU fundida ao fragmento N-terminal da YFP) e LUH-pSPYCE (SEU fundida ao fragmento N-terminal da YFP), ambos muito grande para a difusão para o núcleo, contêm sinais de localização nuclear 2,5. O sinal detectado YFP fluorescente no núcleo da célula da cebola (Figura 1 B, E) indica que SEU-pSPYNE e LUH-pSPYCE podem interagir em núcleos de cebola. Nenhum sinal fluorescente é detectado em cebolas bombardeados com mix de pSPYNE plasmídeo vetor plasmidial contendo o fragmento N-terminal da YFP e LUH-pSPYCE (Figura 1C, F). O BiFC mediada YFP fluorescência é significativamente mais fraca do que a fluorescência da GFP-mediada da 35S:: GFP plasmídeo. Em nosso estudo, eles também mostraram localização subcelular diferente (compare com a Figura 1A B). Além disso, o número de células fluorescentes é significativamente menor para BiFC, como BiFC só funciona quando ambos os plasmídeos parceiro entrar com sucesso e são expressos nas células da cebola mesmo. <img src="/files/ftp_upload/1963/1963fig1.jpg" alt="Figura 1" /> Figura 1. Fluorescente (A, B, C) ​​e de campo claro (D, E, F) imagens de células de cebola bombardeados com diferentes construções de plasmídeos. (A) e (D), as células da cebola bombardeados com 35S:: GFP. GFP difunde por todo o citoplasma eo núcleo. (B) e (E), as células da cebola bombardeados com igual mistura molar de 35S:: SEU-pSPYNE (SEU fundida ao fragmento N-terminal da YFP) e 35S:: LUH-pSPYCE (LUH fundida ao fragmento C-terminal ) DNA plasmidial. Uma interação entre SEU-pSPYNE e LUH-pSPYCE é mostrado por um núcleo fluorescente. (C) e (F), um controle negativo mostrando células de cebola bombardeados com misturas de vetor pUC19-SPYNE e LUH-pSPYCE plasmids. Nenhum sinal fluorescente é detectado.

<ol>
	<li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9 (4), 789-789 (2002).</li><li>Sitaraman, J., Bui, M., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LEUNIG_HOMOLOG+and+LEUNIG+perform+partially+redundant+functions+during+Arabidopsis+embryo+and+floral+development.">LEUNIG_HOMOLOG and LEUNIG perform partially redundant functions during Arabidopsis embryo and floral development.</a> Plant Physiol. 147 (2), 672-672 (2008).</li><li>Walter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant J. 40 (3), 428-428 (2004).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li><li>Azhakanandam, S., Nole-Wilson, S., Bao, F., Franks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEUSS+and+AINTEGUMENTA+mediate+patterning+and+ovule+initiation+during+gynoecium+medial+domain+development.">SEUSS and AINTEGUMENTA mediate patterning and ovule initiation during gynoecium medial domain development.</a> Plant Physiol. 146 (3), 1165-1165 (2008).</li><li>Zhang, X. N., Mount, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+alternatively+spliced+isoforms+of+the+Arabidopsis+SR45+protein+have+distinct+roles+during+normal+plant+development.">Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.</a> Plant Physiol. 150 (3), 1450-1450 (2009).</li></ol>

Não há conflitos de interesse declarados.

<ol><li> Para usuários de primeira vez, recomendamos a prática de todo o processo, desde a preparação do cartucho para observação, com um plasmídeo repórter única constitutiva, como 35S:: GFP ou 35S:: GUS. Usamos um 35S:: GFP plasmídeo obtido a partir de drs. Steve Monte e Zhang Xiaoning 6 (Figura 1A, D). O gene cartuchos de tiro controle vendido a RioRad (Cat 165-2244) não são adequados para as plantas. </li><li> Para BiFC, um segundo controle positivo é necessário, que deve ser composto de dois conhecidos parceiros in...

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<td>Yellow onion</td>
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<td>Any supermarket</td>
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<td>Helios Gene Gun</td>
<td>&nbsp;</td>
<td>BioRad</td>
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<td>Fluorescent microscope with appropriate filters for GFP or YFP</td>
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bimolecular fluorescence complementation (bifc) assay for protein-protein interaction in onion cells using the helios gene gun

Investigação da função dos genes em diversos organismos baseia-se no conhecimento de como os produtos de genes interagem uns com os outros em seu ambiente normal de celular. O Bimolecular Fluorescência de Complementação Assay (BiFC)<sup&gt; 1</sup&gt; Permite aos pesquisadores visualizar interações proteína-proteína em células vivas e se tornou uma ferramenta de pesquisa essencial. Este ensaio é baseado na associação facilitada de dois fragmentos de uma proteína fluorescente (GFP), que são cada fundido a um parceiro em potencial interação de proteínas. A interação dos dois parceiros proteína facilitaria a associação da N-terminal e C-terminal de fragmento de boas práticas agrícolas, conduzindo a fluorescência. Para os pesquisadores células vegetais, cebola epidérmicas são um sistema ideal experimental para a realização do ensaio BiFC por causa da facilidade na obtenção e preparação de tecidos e cebola a visualização direta de fluorescência com fluorescência de fundo mínimo. O Gene Gun Helios (BioRad) é comumente usado para bombardear DNA plasmídeo em células de cebola. Nós demonstramos o uso de Helios Gun Gene introduzir constrói plasmídeo por dois interagindo<em&gt; Arabidopsis thaliana</emFatores&gt; transcrição, Seuss (SEU) e homólogo Leunig (LUH)<sup&gt; 2</sup&gt; Ea visualização de suas interações mediadas por BiFC em células epidérmicas de cebola.

Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun at JoVE.com

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

Este artigo ilustra como usar corretamente o BioRad Helios Gene Gun para introduzir DNA plasmidial em células epidérmicas de cebola e como testar para interações proteína-proteína em células de cebola com base no princípio de Complementação de fluorescência Bimolecular (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 ...

bimolecular-fluorescence-complementation-bifc-assay-for-protein

Research

JoVE Journal

Biology

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

19.5K visualizações.  University of Maryland. Este artigo ilustra como usar corretamente o BioRad Helios Gene Gun para introduzir DNA plasmidial em células epidérmicas de cebola e como testar para interações proteína-proteína em células de cebola com base no princípio de Complementação de fluorescência Bimolecular (BiFC)

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

Gene-gun Transfection of Hippocampal Neurons

Gene-gun Transfection de neurônios do hipocampo

Biologia

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparação de Gene Gun Bullets e Transfection biobalística de neurônios em cultura Slice

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

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.

Complementação de fluorescência bimolecular

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

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

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

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

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Detecção de interações proteína na planta através de um gateway de Complementação de fluorescência Compatível Bimolecular (BiFC) Sistema

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

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 ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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.

Citometria de fluxo e de Complementação de fluorescência Bimolecular: A alta taxa de transferência método quantitativo para estudo de interação proteína-proteína

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.

Interações proteína-proteína visualizado por Bimolecular Fluorescência de Complementação em tabaco Protoplastos e folhas

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.

Investigando interações proteína-proteína em células vivas usando a transferência de Bioluminescência Resonance Energy

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imagens de fluorescência com um nanômetros Precisão (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...