Agradecemos por nos enviar EUROSARF levedura TAP plasmídeos de expressão. Nós também estamos gratos pela ajuda editorial de Ryan Labadens. Este trabalho foi apoiado por uma bolsa do NIH / NINDS (R01NS070962) para CW

1. Gerar UAS-marcado pela TAP Moscas transgênicas <ol><li> Gerar pUAST marcadas pela TAP construções de DNA. <ol><li> Decidir qual o lado (N-ou C-terminal) da proteína isco a etiqueta TAP deverá ser fundido a, com base na estrutura / função da proteína. Veja a discussão para mais detalhes. </li><li> Subclonar a região de cDNA codificadora do gene de interesse para os múltiplos locais de clonagem (MCS) dos vectores de pUAST-NTAP ou pUAST-CTAP para gerar N-ou C-terminal marcadas transgenes UAS-TAP, respectivamente. Veja na Figura 1 mapas detalhados e sítios de restrição utilizáveis ​​e quadros de leitura. </li></ol></li><li> Gerar UAS-marcado-TAP voa transgênicos. <ol><li> Gerar moscas transgênicas seguindo protocolos padrão, utilizando P-mediada pelo elemento de inserção 11. Um certo número de serviços de injecção estão disponíveis comercialmente. </li><li> Atravessar neuronal condutor de GAL4 (ou seja BG380-GAL4) para cada linha transgénica individual e determinar os níveis de expressão de proteínasde cada linha por Western blot (com anticorpo anti-peroxidase Peroxidase) e / ou a imunomarcação (com anticorpo anti-TAP). Em geral, recomenda-se uma linha transgénica com um nível de expressão de proteína que é próximo da proteína endógena para procedimentos TAP. Veja a discussão para mais detalhes. </li><li> PerformGAL4/UAS-based experiências de socorro para confirmar a funcionalidade dos transgenes marcadas com TAP, se a perda de função dos genes mutantes de interesse estão disponíveis. Escolha um transgene que pode salvar substancialmente os fenótipos mutantes para os seguintes experimentos TAP. </li></ol></li></ol> 2. Preparar amostras para a TAP Procedimento <ol><li> Criar um stock de mosca que transporta um controlador neuronal GAL4 (por exemplo BG380-Gal4) e o transgene marcado com TAP escolhido, a fim de facilitar a ampliação de amostras de moscas. Recolha as progênies F1 do piloto GAL4 ea cruz UAS-transgene em casos raros, quando a combinação acima faz com que a sobrevivência eo crescimento desvantagem. &lt;/ Li&gt; <li> Coletar amostras de pequena escala e otimizar a condição de lise para solubilizar a proteína marcadas-TAP. <ol><li> Fazer uma série de tampões de lise usando uma combinação de detergentes não iónicos de NP-40 (0,1-1%), NaDOC (0,1-1%) e Triton X-100 (0,05-0,5%). Ver Tabela 1 e discussão para mais informações. </li><li> Em cima de um bloco de CO2, use # 5 fórceps para dissecar 20 cabeças adultas do transgene TAP expressar moscas e recolher em um tubo de 1,5 ml para testar cada condição de tampão de lise. </li><li> Adicionar tampão teste de lise 100 ul para o tubo e homogeneizar as cabeças acariciando cima e para baixo com um pilão de plástico, em seguida, adicione um outro tampão teste 100 ul. </li><li> Girar o lisado cabeça em 21.500 xg durante 10 minutos (4 ° C), e separar do sobrenadante e sedimento, após a centrifugação. Adicionar 25 ul de tampão de carregamento 2 x SDS ao precipitado e tampão de carregamento de 10 ul 4x SDS a 30 ul de sobrenadante, respectivamente. </li><li> Ferver as duas amostras durante 5 minutos e analisam-los lado-Lado utilizando SDS-PAGE e subsequente Western Blot com anticorpo PAP. Determinar a solubilidade pela relação dos níveis de proteína-TAP em sobrenadante contra o sedimento. </li></ol></li><li> Prepare amostra em grande escala <ol><li> Expandir e recolher moscas adultas. <ol><li> Expandir as ações neuronal-GAL4/UAS-TAP-transgene em garrafas e virar as garrafas a cada 3 dias até acumulativas 250 garrafas são usados ​​para a coleção. </li><li> Colete de 1-3 dias de idade adulta voa para 50 ml tubos cônicos, coloque o tubo em nitrogênio líquido imediatamente para congelador as moscas. Armazenar as moscas em um congelador a -80 ° C. Note-se que o volume das moscas não deve ser superior a 2/3 do tubo de 50 ml. </li></ol></li><li> Colete cabeças mosca (executar este passo em cima de gelo seco em pó). <ol><li> Retire as peneiras prechilled eo almofariz e pilão do freezer -80 ° C e colocá-los em gelo seco, de preferência dentro de um grande balde de gelo. Empilhe duas peneiras de teste padrão dos EUA com um No. 25 no the superior e n ° 40, na parte inferior. </li><li> Pegue as moscas congelados para fora e deixá-los em nitrogênio líquido e manter as moscas lá por cerca de 10 min. Vortex ou agitar os tubos vigorosamente para quebrar as cabeças, pernas e asas dos corpos. </li><li> Despeje a mistura para o peneiro superior e, em seguida, agitar as peneiras vigorosamente, mantendo ambas as peneiras juntos. Depois de peneirado, os corpos vão ficar no topo peneira, voar cabeças serão retidos na peneira de fundo, e as pernas, asas e outros detritos vão cair para o gelo seco. Separe as duas peneiras e transferir cuidadosamente as cabeças de voar para a argamassa frio. </li></ol></li><li> Homogeneizar as cabeças de mosca <ol><li> Em cima do gelo seco, moer a cabeça com a argamassa eo pilão para partículas de pó, em seguida, transferir o pó a 15 ml de vidro Dounce Grinder tecido que foi previamente resfriada em gelo. </li><li> Meça os pesos do moedor de antes e depois da amostra cabeça foi derramado nele, e em seguida, calcular o quanto o samp cabeçale pesa. Um total de 6-15 gramas de cabeças mosca será suficiente para cada experimento TAP ajuste de acordo com os níveis de expressão da proteína. Adicionar 15 ml de tampão de homogeneização gelada (lise otimizado no passo 2.2) para o pó e, em seguida, derrame com o grande pilão folga até que seja fácil para os pilão para ir para cima e para baixo. Manter o moinho de vidro em gelo em todos os momentos. </li></ol></li><li> Prepare o sobrenadante para a TAP <ol><li> Transferir o homogeneizado para um tubo de centrifugação de alta velocidade de rotação e durante 20 min a ~ 50.000 xg (4 ° C). Transferir o sobrenadante para um novo tubo de centrifugação de alta velocidade e repetir a centrifugação mais uma vez. </li><li> Transferir o sobrenadante para um tubo de ultracentrifugação e executar um 40 min ~ 250.000 xg rotação para limpar ainda mais o sobrenadante. O sobrenadante está pronto para os procedimentos de purificação por afinidade em tandem após ultracentrifugação. </li></ol></li></ol></li></ol> 3. TAP Purificação </p&gt; As secções que se seguem foram obtidos a partir do laboratório de TAP Séraphin protocol 12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf ) <ol><li> Realize IgG talão purificação por afinidade <ol><li> Prepare IgG Sepharose talão enquanto as amostras são centrifugadas. Lavar 3x 400 ul de contas de IgG num tubo Falcon de 15 ml com 10 ml de tampão de lavagem frio IgG. Para cada lavagem, balançar suavemente o tubo para 2 min, e depois girar as contas a 1.000 xg por mais 2 min. No final da terceira lavagem, remover o tampão e deixar apenas as esferas no tubo. </li><li> Transferir cuidadosamente o sobrenadante clarificado (~ 15 ml) para o tubo de 15 ml contendo as contas de IgG. Incubar os grânulos e mistura lisado cérebro, a 4 º C em um nutator durante 2 horas. </li><li> Configurar uma coluna micro limpo e vazio com cerca de 15 ml de volume total na câmara fria. Coloque a mistura do grânulo IgG por constantemente despejando a mistura para a coluna, não tentar prender qualqueras bolhas de ar no interior da coluna. Permitir que as contas para se instalar na coluna e no tampão para drenar lentamente por fluxo de gravidade. </li><li> Lavar cuidadosamente a coluna com 10 ml de tampão de lavagem frio IgG depois de todo o lisado cérebro fluiu através da coluna de IgG resolvido. Repita o 2x lavagem. Nota: nunca deixar secar o talão no ar. </li></ol></li><li> Realize TEV clivagem <ol><li> Após a terceira lavagem, lava-se a coluna outra vez com 10 ml de tampão de clivagem de TEV. Esta etapa prepara o talão IgG que seqüestra a complexa isca para TEV clivagem. </li><li> Imediatamente antes da última gota de tampão de TEV é sobre a escorrer para fora, colocar uma tampa na parte inferior da coluna para bloquear o fluxo, adicionar 1,3 ml de tampão de TEV contendo 130 unidades da enzima de TEV para a coluna, e em seguida tampão de forma segura a parte superior do a coluna. Certifique-se de que a coluna é bem fechadas em ambas as extremidades. </li><li> Rodar a coluna a 18 º C durante 2 horas para permitir que a enzima de TEV para clivar o péptido no local da TEV e libertar a proteína whi complexole deixando para trás a proteína de um péptido de domínio ligado a esferas de sefarose da IgG. </li></ol></li><li> Realize Calmodulina talão purificação por afinidade <ol><li> Prepare os grânulos de calmodulina, enquanto os grânulos de IgG foram incubadas com a enzima de TEV. Lavar 200 ul grânulos calmodulina em um Falcon de 15 ml de tubos de 3x, de cada vez com 10 ml de tampão de ligação frio calmodulina. Para cada lavagem, agitar suavemente o tubo durante 2 min num nutator, e depois girar as esferas a 1000 xg durante 2 min. No final da terceira lavagem, retirar todo o tampão e deixar apenas as esferas no tubo. </li><li> No final da incubação TEV (passo 3.2.3), o retorno da coluna IgG de volta para a sala fria e coloque-o em linha reta. Vamos as contas se contentar com 10 min. </li><li> Retire a tampa superior e, em seguida, a tampa inferior e, em seguida, recolher o produto de clivagem 1,3 ml TEV em um tubo Falcon de 15 ml. Deixe o tampão drenar completamente. Adicionar um buffer de 200 mL TEV adicional para a coluna para empurrar para fora o volume morto da coluna, recolher o fbaixa no mesmo tubo. </li><li> Adicionar 4,5 ml de tampão de calmodulina e 4,5 mL de 1 M de CaCl 2 de ligação para o produto de clivagem de 1,5 ml de TEV recolhidos acima. O CaCl 2 serve para dosear o EDTA no buffer de TEV. Transferir a mistura de 6 ml para o tubo contendo as esferas de calmodulina e rodar o tubo a 4 ° C num nutator durante 1 hora. </li><li> Configure outra coluna micro limpo e vazio com cerca de 10 ml de volume total na câmara fria. Coloque a mistura de pérolas para calmodulina para a coluna e permitir que a drenar pela força da gravidade. </li><li> Quando toda a solução fluiu através da coluna de calmodulina resolvido, lava-se a coluna duas vezes, cada uma com 10 ml de tampão de ligação frio calmodulina. Nota: evitar perturbar as contas calmodulina e tentar manter a superfície dos grânulos de tão plana quanto possível, durante a lavagem. </li></ol></li><li> Eluir o complexo de isca de coluna calmodulina. Logo após a lavagem, eluição da coluna de calmodulina com cinco frações de 200 mL frio Calmodutampão de eluição lin. Para cada fracção, adiciona cuidadosamente 200 ul de tampão de eluição à coluna e recolher o eluato com um marcado tubo Eppendorf de 1,5 ml. Repita este 4x. </li><li> Analisar o complexo de proteína por SDS-PAGE <ol><li> Tomar uma pequena alíquota de cada um dos cinco fracções (aproximadamente 30 ul) e adicionar tampão de carga de SDS. Ferver as amostras durante 5 minutos e carregar as amostras de lado-a-lado com os marcadores moleculares de proteínas em um gradiente (4-15%) em gel de SDS-PAGE. </li><li> Após as amostras terem totalmente resolvidas no gel, parar a eletroforese e manchar o gel com todos os procedimentos de coloração à base de L-250 sensíveis Coomassie coloidal como &quot;prata azul &#39;manchando 13. Coloração prata é opcional, mas não preferível, porque não é totalmente compatível com a análise de espectrometria de massa subseqüente. Armazenar o resto do eluato num congelador a -80 ° C para posterior análise, tal como a espectrometria de massa para descobrir as identidades moleculares do complexo de proteína purificada. See discussão. </li></ol></li></ol>

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Método de purificação por afinidade em tandem (TAP) oferece um protocolo de purificação dupla que permite o isolamento e enriquecimento de complexos de proteínas por meio de duas etapas independentes de purificação de afinidade. O design da marca TAP não se restringe ao que é apresentado neste protocolo, outros domínios de ligação de proteínas e motivos também são aplicáveis ​​se as condições do tampão são ajustados em conformidade. Um bom exemplo de outras tags TAP é a marca GS-TAP, uma combin...

Definindo as vias moleculares ou redes que mediam um processo biológico em particular é um dos objetivos finais da pesquisa biomédica. Fly geneticistas têm dependido fortemente de genética para a frente, especialmente modificador telas genéticos (tanto Enhancer e telas supressores), para identificar os fatores que trabalham em conjunto, em paralelo com, ou a montante ou a jusante de um gene de interesse. No entanto, as telas de genética para a frente muitas vezes não conseguem identificar genes essenciais que, quando mutado, causar letalidade em estágios iniciais de desenvolvimento, ou genes com redundância funcional e compensação cuja perda de função só causar defeitos subtis que são difíceis de marcar. Uma maneira de superar essa dificuldade é para triagem de interações proteína-proteína diretos. Por mais de uma década, uma lista crescente de métodos bioquímicos, incluindo levedura de dois híbridos, display de fagos, cross-linking química, Co-IP, Tandem Affinity Purification (TAP), etc. têm sido utilizados para investigar proteína-proteína interações. Cada uma dessas abordagens tem seu próprio conjunto de pontos fortes e fracos em relação à sensibilidade e especificidade. Entre eles, o método permite que a TAP para detecção da interacção física sob condições quase fisiológicas, preserva a especificidade e consistência 2 e inclui a capacidade de estender a análise de alto rendimento de 3,4. O método TAP foi originalmente desenvolvido na levedura por colegas Rigautand 1. Neste método, uma proteína de interesse é expressa com uma etiqueta TAP. A etiqueta TAP abriga dois domínios de ligação de afinidade independentes: uma proteína de um domínio que se liga a IgG e um domínio de ligação à calmodulina. Os dois domínios estão separados por um local de clivagem de TEV (Tobacco Etch Vírus). Essa combinação permite a duas rodadas independentes de purificações de afinidade para reduzir suficientemente ligações inespecíficas e enriquecer vinculações específicas 1. Para este exemplo, o método TAP é um poderoso metodod para identificar interacções in vivo de uma determinada proteína, embora o sobre-expressam a proteína exógena pode torná-lo mais propenso a associar-se com proteínas que normalmente não fazem complexo com o seu homólogo endógeno. Uma vez que o seu desenvolvimento, o método da TAP foi aplicado em muitos outros sistemas, incluindo--base de cultura de células e outros sistemas de 5,6 in vivo, os sistemas modelo de 6-9. Aqui nós descrevemos a adaptação do método TAP em Drosophila. O primeiro gerar pUAST-NTAP e pUAST-CTAP vectores para facilitar a clonagem e a fusão da etiqueta TAP ou o N-ou C-terminal do gene de interesse. A-marcado com TAP UAS transgene é, então, expresso no sistema nervoso sob o controlo de um controlador 10 de GAL4 neuronal. Em seguida, um grande número de cabeças de moscas adultas serão recolhidos, que tem elevado teor de células neuronais e são fáceis de separar a partir de outras partes do corpo, após a congelação com base em diferenças de tamanho. As cabeças adultas são homogeneizados e limpou bcentrifugações sequenciais y, e o sobrenadante é sujeito a um procedimento de TAP descrito abaixo.

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		<tr height="42">
			<td height="42" style="height:42px;width:307px;">U.S.A. standard test sieve No. 25</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-18&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-21</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;Kontes Dounce Tissue Grinders 15 ml</td>
			<td>Kimble Chase</td>
			<td>885300-0015</td>
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			<td height="21" style="height:21px;width:307px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">IgG sepharose beads</td>
			<td>Pharmacia</td>
			<td style="width:119px;">17-0969-01</td>
			<td></td>
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			<td height="21" style="height:21px;">Econo-column 0.7 cm x 20 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4721</td>
			<td></td>
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			<td height="21" style="height:21px;">Econo-column 0.5 cm x 15 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4716</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Calmodulin beads</td>
			<td style="width:104px;">Stratagene</td>
			<td style="width:119px;">214303</td>
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			<td height="21" style="height:21px;">Coors Mortar and Pestle</td>
			<td style="width:104px;">CoorsTek</td>
			<td>60311</td>
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			<td height="21" style="height:21px;">AcTEV Protease</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
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			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:104px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Mix</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

Telas genéticas realizadas utilizando Drosophila melanogaster (mosca da fruta) fizeram numerosas descobertas marco no avanço das ciências biológicas. No entanto, o uso de telas bioquímicas destinadas a estender o conhecimento adquirido a partir da análise genética foi explorada apenas recentemente. Aqui nós descrevemos um método para purificar o complexo de proteínas que se associa com qualquer proteína de interesse a partir de cabeças de moscas adultas. Este método tira vantagem do sistema de Drosophila GAL4/UAS para expressar uma proteína isco fundido com um tag de afinidade em tandem de Purificação (TAP) em neurónios mosca in vivo, e, em seguida, executa duas rondas de purificação utilizando um procedimento de TAP semelhante à originalmente estabelecido em levedura 1 para purificar o complexo de proteína que interage. No final deste procedimento, uma mistura de vários complexos de proteína é obtido cujas identidades molecular pode ser determinada por espectrometria de massa. Validação das proteínas candidatos serão beneficiados com a resource e facilidade de realizar estudos de perda de função em moscas. Abordagens semelhantes podem ser aplicados a outros tecidos de moscas. Acreditamos que a combinação de manipulações genéticas e esta abordagem proteômica no sistema modelo mosca tem um tremendo potencial para a resolução dos problemas fundamentais no campo da neurobiologia e além.

Aqui demonstramos o nosso esforço na identificação de proteínas Highwire-que interagem no cérebro da mosca. Highwire (Hiw) e seus homólogos de vertebrados e invertebrados são enormes ligases de ubiquitina que regulam o desenvolvimento e reparação do sistema nervoso 14. Eles compartilham um número de domínios funcionais altamente conservadas. No entanto, suas ações moleculares não são totalmente claras. Trabalho feito em verme, mosca e do rato levou à do modelo de trabalho actual que funciona c...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP at JoVE.com

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila é famoso por sua poderosa manipulação genética, mas não para a sua adequação de análises bioquímicas em profundidade. Aqui apresentamos um procedimento baseado em TAP para identificar interagindo parceiros de qualquer proteína de interesse a partir do cérebro da mosca. Este procedimento pode potencialmente levar a novos caminhos de pesquisa.

Identificando interacção proteína-proteína em<em&gt; Drosophila</em&gt; Chefes Adulto por Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

identifying-protein-protein-interaction-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

13.8K visualizações.  Louisiana State University Health Sciences Center.  Drosophila é famoso por sua poderosa manipulação genética, mas não para a sua adequação de análises bioquímicas em profundidade. Aqui apresentamos um procedimento baseado em TAP para identificar interagindo parceiros de qualquer proteína de interesse a partir do cérebro da mosca. Este procedimento pode potencialmente levar a novos caminhos de pesquisa. 

Vídeo: Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificação de Proteínas ortogonais Facilitado por um Tag Affinity Pequenas Bispecific

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Biologia

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificação de parceiros de proteína Interagir utilizando Tandem purificação por afinidade

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento de cromatina por purificação de RNA (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purificação de Transcrições e metabolitos de<em&gt; Drosophila</em&gt; Cabeças

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Método de purificação de proteína livre de Determinação Affinity Binding por termoforese Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

A identificação da proteína-proteína Locais de interacção Usando Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

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 ...