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...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</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>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
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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 ...

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Biology

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

17.2K 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

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

Foto-Induzida Cross-Linking de Proteínas Unmodified (picup) Aplicada à Peptídeos amiloidogênicas

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

Biologia

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificação de complexos de proteínas com proteômica quantitativa em S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizando Único complexos moleculares In Vivo Usando microscopia de fluorescência avançada

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME Método (Enzyme Linked Immuno Magnetic eletroquímica) para Detecção de micotoxinas

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Na imagem in vivo de larvas Drosophila Intact no Sub-celular Resolução

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Calorimetria isotérmica de titulação para medição de Macromoléculas-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

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 ...

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 ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Mapeamento baseado em AFM das propriedades elásticas de paredes celulares: em tecido, os celulares, e as Resoluções Subcelulares

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

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) ...