Este trabalho foi financiado por subvenções dos Institutos Nacionais de Saúde para VF (NS42599 e GM077569).

1. Preparando-se para Crosslinking <ol><li> Você vai precisar de placa de um número suficiente de células para permitir o isolamento de 500 mg de proteína por reação de tubo padrão. Um único tubo pode ser suficiente para a identificação de supostos interatores de uma proteína de interesse por immunoblot. Neste caso, o material ligado talão pode ser eluída com SDS-PAGE tampão de amostra (imunoprecipitação). Para análise por espectrometria de massa, o número de reações padrão deve ser aumentado, pelo menos, dez vezes e complexos de proteína deve ser eluída por a concorrência com um antígeno de peptídeo reconhecido pelo anticorpo utilizado (cromatografia de imunoafinidade). Esta estratégia vai permitir o isolamento de complexos de proteínas livres de imunoglobulina. </li><li> A densidade celular ideal para a reticulação é entre 75% e 90% confluência. Em condições de alta confluência, as células tendem a se acumular uns sobre os outros, o que diminui a acessibilidade ao reticulador celular permeável a todas as células. </li><li> Tipo de célula e condição experimental deve ser colocado na parte inferior da placa celular. Rotineiramente incluir controlos com veículo sozinho. </li><li> Prepare todas as soluções EXCETO a solução DSP antes de reticulação. <ol><li> Prepare um estoque de tampão fosfato-salina tamponada com CaCl 2 0,1 mM e CaCl 2 1 mM (PBS / Ca / Mg) antes de iniciar o experimento. A adição de íons de cálcio e magnésio é fundamental para a adesão das células à placa de cultura durante o curso do experimento. Armazenar a 4 ° C. </li><li> 50X Cocktail Inibidor da Protease Completa - 1 tablete dissolvido em 1 ml de água Milli-Q. Armazenar a -20 ° C. </li><li> 20% Triton X-100 Pesar 10g Triton X -100 e diluir em volume total de 50 ml de água Milli-Q. Rocha a 4 ° C durante a noite e armazenar a 4 ° C até o uso. Não armazenar por mais de um mês. Use diluições deste estoque de 20% para preparar o buffers lise e IP descrito abaixo. </li><li> 50X Quenching DSP solução 1M de TRIS pH 7.4. Conservar à temperatura ambiente. </li><li> Preparar um tampão 10x uma solução. Tampão 10X A: <ul><li> 50 mL 1 M HEPES </li><li> 150 mL de NaCl 5 M </li><li> 10 mL 0,5 M EGTA </li><li> 250 L 2 M MgCl 2 </li><li> Ajustar o pH para 7,4 </li><li> Trazer um volume final de 500 mL </li></ul> O tampão 10X A solução é diluída em tampão 1X A necessária como. 1x tampão A também é usado em lisado e-magnético-imuno precipitação buffers. Um buffer 1X <ul><li> 10 mM Hepes </li><li> 150 mM NaCl </li><li> 1 mM EGTA </li><li> 0,1 mM MgCl 2 </li><li> pH 7,4 </li></ul></li><li> Lise Tampão Tampão 1X A + 0,5% Triton X-100. </li><li> Imuno-precipitação Magnetic buffer (Buffer IP) 1X tampão A + 0,1% Triton X-100. </li></ol></li></ol> 2. Preparar a Solução Crosslinking <ol><li> Prepare a solução DSP imediatamente antes de aplicar para as células. DSP é altamente hidrofóbico e deve ser dissolvido em DMSO antes de se diluir em PBS / Ca / Mg buffer. Dissolver 40 mg de DSP em 1 mL de DMSO. Isso faz com que uma solução de 100 mM de DSP (O peso molecular de DSP é 404,42 g / mol). </li><li> Quente um volume adequado de PBS / Ca / Mg a 37 ° C, a fim de facilitar a diluição do DSP / DMSO em PBS. Volumes necessários para tamanhos variados placa: 6-bem placa 2 mL por poço 10 centímetros placa 10 mL por placa 15 centímetros placa 20 mL por placa </li><li> Adicionar 10μL de DSP / DMSO solução estoque para cada 1 mL de PBS quente / Ca / Mg. Adicionar DSP / DMSO gota a gota, solução estoque com mistura repetido até que todos os DSP se dissolveu. Faça uma solução de controle de 10 DMSO mL adicionados a cada 1 mL de PBS / Ca / Mg. </li><li> Coloque todas as soluções de PBS / Ca / Mg em um banho de água gelada não superior a 10 min. </li></ol> 3. Prepare Células para Crosslinking <ol><li> Prepare um banho de água gelada que vai caber todas as placas de reticulação ou incubação controle do veículo. </li><li> Pegue pratos da incubadora de 37 ° C e colocá-los imediatamente para o banho de água gelada. </li><li> Lave as células duas vezes com PBS gelado / Ca / Mg. Use o mesmo volume que você irá usar para a incubação reticulador (ver secção 2.2). </li><li> Remover segunda lavagem e adicionar qualquer controle do veículo ou DSP solução tampão de reticulação. </li><li> Incubar no gelo durante duas horas. Você deve verificar as placas aproximadamente a cada 20 minutos para garantir que todas as células estão cobertas pela solução. Você pode notar uma pequena quantidade de DSP precipitar para fora da solução. Isso é normal. </li></ol> 4. Inativação de Reação DSP <ol><li> Prepare uma solução de têmpera 1X DSP de 20 mM Tris pH 7,4 em PBS / Ca / Mg (20 mL de 1 M Tris pH 7,4 para cada 1 mL de PBS / Ca / Mg). </li><li> Remova o controle do veículo e soluções de reticulação. </li><li> Adicionar gelada solução inativação e incubar no gelo por 15 minutos. </li></ol> 5. Lise celular <ol><li> Durante a incubação DSP quenching, preparar o tampão de lise celular de 0,5% Triton X-100 em tampão Um coquetel inibidor de protease com Completo. Adicionar 20 mL de solução estoque 50X de completas para cada 1 mL tampão de lise. </li><li> Após a incubação 15 minutos quenching DSP, lave as células duas vezes com PBS / Ca / Mg. </li><li> Adicionar tampão de lise + Completo para células e rock a 4 ° C por 30 minutos. Alguns sugeriram volumes de tampão de lise para tamanhos variados placa: <ul><li> 6-bem placa 0,5 mL por poço </li><li> 10 centímetros placa 1 mL por placa </li><li> 15 centímetros placa 1 mL por placa </li></ul></li><li> Após a lise, use um raspador de celular para coletar as células da placa. Certifique-se de manter lisados ​​no gelo para minimizar a atividade de protease </li><li> Lisados ​​de células giro por 15 minutos a 4 ° C em uma bancada de mini-centrífuga em alta velocidade (15.000 xg). </li><li> Pipetar o sobrenadante para um novo tubo. Descartar o pellet. </li><li> Analisar os níveis de proteína e prosseguir com imunoprecipitação. </li></ol> 6. Preparando Beads Imuno-magnética Precipitação Nota: Esta etapa é geralmente iniciado diretamente após o início da incubação de reticulação duas horas. <ol><li> Combine 30 mL de Dynal esferas imunomagnéticas, 500 mL de tampão de IP (1x tampão A + 0,1% Triton X-100) e a quantidade adequada de antígeno-anticorpo específico para screwtop tubos de microcentrífuga. Prepare-anticorpo livre e tubos de anticorpos não-específicas dos controlos negativos. A quantidade adequada de anticorpos deve ser determinado com base em antígenos individuais durante experimentos separados. Rotular todos os tubos de IP. </li><li> Permitir que os tubos de IP para incubar em um rocker end-over-end em temperatura ambiente por duas horas. Alternativamente, incubar os tubos IP com anticorpo overnight a 4 ° dia anterior ao crosslinking. </li><li> Após a incubação, fazer um rápido 10 segundo mini-centrifugação para coletar as esferas na parte inferior do tubo. </li><li> Deslize os tubos no suporte magnético, que vai puxar a esferas magnéticas longe do fundo do tubo. Com as contas em segurança para fora do caminho, agora você pode facilmente lavar qualquer anticorpo não ligado. </li><li> Use uma ponta do aspirador pequeno furo, como uma ponta de carga gel, ou p200 dica para remover o tampão de incubação e qualquer anticorpo não ligado. Assim como o volume de incubação é removido adicionar 1 mL de tampão de IP. </li><li> Tampe o tubo, delicadamente ressuspender as esferas, e incubar todos os tubos IP em temperatura ambiente por 5 minutos com fim-over-final da rotação. </li><li> Repita o passo de lavagem de 5 minutos (passos 6,3 por 6,6) mais uma vez com um mililitro de tampão fresco IP. </li><li> Os tubos de IP pode permanecer na incubação, lavar durar até o lisado reticulado está pronto para ser adicionado às esferas. </li></ol> 7. Incubar Lysate Crosslinked com Imuno-magnética Beads <ol><li> Após a última rodada lavar imuno-magnética contas, para baixo em uma mini-centrífuga por 10 segundos. Em seguida, proceder a deslizar os tubos no suporte magnético. </li><li> Mais uma vez, usando uma ponta de aspiração pequeno furo, retire o volume de incubação do tubo. Imediatamente adicionar 500 mL de lisado celular reticulado (assumindo lisado a 1 mg / mL) para os tubos contendo imuno-magnética contas. Se você estará usando competição peptídeo como controle negativo, uma quantidade adequada de peptídeo deve ser incluída neste momento. Experiências anteriores devem ser realizados para determinar as condições de concorrência adequadas peptídeo. Alternativamente, você pode querer ter lisado livre imuno-magnética contas como controle negativo. Neste caso, adicionar 500 mL de imediato de Lysate Buffer (1x tampão A + 0,5% Triton X-100) + completo para as contas imuno-magnética. </li><li> Delicadamente ressuspender as esferas. Grânulos em suspensão devem ser incubadas a 4 ° em uma rotação fim-over-end por 2 horas. </li></ol> 8. Lavar Lysate Unbound de Contas <ol><li> Uma vez lisado teve tempo de incubação suficiente, faça uma rotação rápida 10 segundo de uma mini-centrífuga. Em seguida, deslize os tubos no suporte magnético. O suporte magnético deve ser mantida em banho de gelo para o restante do experimento. </li><li> Usando um pequeno furo ponta do aspirador, remova todos os lisado desacoplado. Imediatamente depois de tudo lisado não ligado é removido adicionar 1 mL de tampão de IP. </li><li> Tampe o tubo e ressuspender delicadamente as contas. </li><li> Uma vez que as contas são ressuspenso repita o passo de revestimento de esferas (8.1). </li><li> Novamente aspirar fora de todos o volume de incubação e imediatamente adicionar 1 mL de tampão de IP, então a tampa do tubo. </li><li> Delicadamente ressuspender as contas em Tampão IP. Incubar grânulos em suspensão por 5 minutos a 4 ° com fim-over-end de balanço. </li><li> Repita os passos talão de lavar roupa (8,4 por 8,6) 4 vezes mais. </li></ol> 9. Desnaturar Amostra e Coleta de Contas <ol><li> Depois de todos os tubos de imuno-magnética precipitação foram lavados, re-sedimento as esferas (8.1) e tubos de deslize para osuporte magnético. </li><li> Proteínas vinculados às esferas imuno-magnéticos são removidos em condições de desnaturação. Aspirar fora do tampão de lavagem última IP e remova o tubo de IP do suporte magnético. </li><li> Adicionar 1x tampão de amostra para o tubo de Gel IP logo acima do pellet de contas para as contas piscina na parte inferior do tubo novamente. A quantidade de tampão de amostra depende do tamanho bem do gel a ser utilizado. Você pode precisar bata suavemente o fundo do tubo para obter todas as contas ressuspenso no tampão de amostra Gel. </li><li> Repita o processo de desnaturação (9,2 por 9,3) para cada tubo de IP. </li><li> Imuno-magnética contas ressuspenso em tampão de amostra Gel são, então, aquecido a 75 ° C por 5 minutos para completar o processo de desnaturação. </li><li> Uma vez que a amostra foi desnaturado que pode ser executado em um gel SDS-PAGE para secar ocidental. Esvaziado Imuno-Magnética Beads pode ser re-peletizada por centrifugação e retirado da amostra com o suporte magnético. </li></ol> 10. Eluição dos Complexos Crosslinked com peptídeos antigênicos (cromatografia de imunoafinidade). <ol><li> Depois de todos os tubos de imuno-magnética imunoprecipitação foram lavados, re-sedimento as esferas (8.1) e tubos de deslize no suporte magnético. Aspirar o sobrenadante e adicionar 10 ml de tampão A suplementado com o peptídeo antigênico reconhecido pelo anticorpo utilizado para immunoisolation de complexos de proteínas. Você deve testar concentrações que variam de 10-200 M para eluição eficiente. </li><li> Incubar as contas por 2 horas a 4 ° C. </li><li> Coloque contas no suporte magnético e cuidadosamente recuperar os 10 mL do material eluído. Salvar este sobrenadante. </li><li> Lavar rapidamente as contas com tampão A e descartar a lavagem. Salvar as contas. </li><li> Adicionar tampão de amostra Gel para o sobrenadante guardado e contas a uma concentração de 1X. Incubar os tubos a 75 ° C por 5 minutos. </li><li> Analisar contas e eluato peptídeo por SDS-PAGE. O eluído deve ser livre de IgG tanto por proteína mancha de SDS-PAGE ou por Immunoblots (Figura 1B). Este material livre de IgG é adequado para espectrometria de massa. </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento de AP-3 complexos de proteínas que interagem e proteínas da membrana. HEK293 células (A) ou células PC12 (B) foram tratados na presença de controle de veículo (DMSO, ímpar pistas Figura 1A) ou DSP (mesmo pistas fig. 1A ou todos os faixas em Fig 1b). Extratos clarificados foram incubadas ou com contas sozinho (Fig. 1A, Lanes 3-4), anticorpos de receptor de transferrina (Fig. 1A, Lanes 5-6), ou AP-3 anticorpos δ (Fig. 1A, Lanes 10/07; Fig. . 1B, faixas 1-10). Imunocomplexos foram eluídos com SDS-PAGE tampão de amostra (Fig. 1A), Buffer A sozinho ou (Fig. 1B, faixas 1-2), ou tampão A suplementado com concentrações crescentes do peptídeo antigênico δ correspondente aos aminoácidos 680 710 dos direitos humanos δ-adaptin (NCBI: AAD03777; gi: 1923266) (Fig. 1B, faixas 3-10). Este peptídeo se liga o anticorpo δ. Após eluição peptídeo, sobrenadantes (S) e esferas (B) foram analisados ​​por immunoblot (Figura 1B). AP-3, que é detectado com anticorpos contra o δ, β3 e σ3 subunidades, coprecipitates com os seguintes fatores solúveis: cadeia pesada clatrina (CHC), o BLOC-1 pallidin subunidade, eo HOPS vps33b subunidade complexo, bem como proteínas da membrana fosfatidilinositol-4-quinase tipo II alfa (PI4KIIα) e que o transportador de zinco 3 (ZnT3). Observe a ausência de cadeias de IgG do mouse pesados ​​no sobrenadante eluído peptídeo na fig. 1B.

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP, uma membrana permeável, reticulador quimicamente redutível com um braço espaçador de 12 Å é usado para estabilizar as interações proteína transitória 1,2,3,4. Aqui nós exemplificou essa estratégia com o complexo adaptador de AP-3 um complexo de proteínas solúveis que reconhece e classifica as proteínas da membrana em vesículas de endossomos 5. AP-3 se liga ao transportador de zinco ZnT3 e os lipídios quinase fosfatidilinositol-4-quinase tipo II alpha, mas não receptor de tran...

<table><tbody><tr><td>Phosphate Buffered Saline</td><td>Invitrogen</td><td>P4417</td><td>Dissolve 1 tablet in 200 mL water; add MgCl&lt;sub&gt;2&lt;/sub&gt; to a final concentration of 1 mM and CaCl&lt;sub&gt;2&lt;/sub&gt; to a final concentration of 0.1 mM</td></tr><tr><td>Dithiobis (succinimidyl propionate) (DSP)</td><td>Thermo Fisher Scientific, Inc.</td><td>22585</td><td>Moisture sensitive, store in air tight container 4&amp;#x00B0;C</td></tr><tr><td>Dimethyl sulphoxide (DMSO) Hybri-Max</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Triton X-100, SigmaUltra</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Dynabeads, Sheep anti-Mouse IgG</td><td>Invitrogen</td><td>110.31</td><td>Beads are also available as sheep anti-rabbit</td></tr><tr><td>Dyna-Mag-2 magnet</td><td>Invitrogen</td><td>123-21D</td><td></td></tr></tbody></table>

isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

A natureza dinâmica de máquinas celular é freqüentemente construída sobre associações de proteína transitória e / ou fraco. Essas interações de baixa afinidade impede métodos rigorosos para o isolamento e identificação de redes de proteínas em torno de uma proteína de interesse. O uso de reticuladores química permite a estabilização seletiva das interações lábil, ignorando assim as limitações bioquímicas para a purificação. Aqui apresentamos um protocolo favorável para células em cultura que usa um reticulador homobifunctional com um braço espaçador de 12 Å, dithiobis (succinimidyl proprionate) (DSP). DSP é clivada pela redução de uma ligação de dissulfeto presentes na molécula. Cross-linking combinada com cromatografia de imunoafinidade de proteínas de interesse com esferas magnéticas permite o isolamento de complexos de proteínas que, do contrário não resistiria a uma purificação. Este protocolo é compatível com técnicas regulares western blot e pode ser ampliada para identificação de proteínas por espectrometria de massa 1. Stephanie A. Zlatic e Pearl V. Ryder contribuíram igualmente para este trabalho.

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

A célula permeável reticulador DSP [dithiobis (succinimidyl propionato)] estabiliza interações transitória e instável<em&gt; In vivo</em&gt;, Que permite o seu isolamento usando técnicas de purificação de proteínas rigorosos complexo. Aqui nós apresentamos uma técnica para as células cultivadas em cultura de reticulação seguido pelo isolamento de complexos de proteínas por imunoprecipitação.

Isolamento de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

isolation-labile-multi-protein-complexes-vivo-controlled-cellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

17.3K visualizações.  Emory University. A célula permeável reticulador DSP [dithiobis (succinimidyl propionato)] estabiliza interações transitória e instável In vivo, Que permite o seu isolamento usando técnicas de purificação de proteínas rigorosos complexo. Aqui nós apresentamos uma técnica para as células cultivadas em cultura de reticulação seguido pelo isolamento de complexos de proteínas por imunoprecipitação.

Vídeo: Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Proteína de captura por afinidade complexos a partir de células de mamíferos Cryomilled

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Bioquímica

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Multímero-PAGE: Um método para capturar e Solução de proteína em amostras biológicas Complexos

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolver purificada por afinidade complexos de proteínas por Azul Native PAGE e Proteína Correlação Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Avaliação de interações proteína-proteína usando uma técnica de digestão na membrana

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Imunologia e Infecção

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 ...

Biologia

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Abordagens genéticas e bioquímicas para<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; Avaliação de oligomerização da proteína: A Case Study rianodina Receptor

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

Combinando reticulação química e espectrometria de massa de complexos de proteínas intactas para estudar a arquitetura de montagens de proteínas com multi- subunidades

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Correção rápida in Vivo e isolamento de complexos translacionais de células eucarióticas

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Isolamento de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

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