Compatível com o DA034783 NIH / NIDA. Agradecemos Bryan A. Killinger de assistência técnica com o multimero-PAGE.

1. Preparação do Tecido <ol><li> Preparar 10 ml de 4x tamp de amostra BN-PAGE (Bis 200 mM (2-hidroxietil) metano amino-tris (hidroximetil) (Bis-Tris), NaCl 200 mM, 40% w / v de glicerol, 0,004% de Ponceau S, pH 7,2 ). NOTA: esta solução pode ser feita com antecedência e guardadas a 4 ° C. </li><li> Dilui-se 250 uL de tampão de amostra 4x em 750 uL de dH2O contendo mistura de inibidores de protease comercial 1x. Vortex e frio no gelo. </li><li> Homogeneizar 20 mg de tecido alvo no tampão de amostra de 1 mL de 1x gelada BN-PAGE com 30 movimentos de um homogeneizador Dounce limpo. NOTA: Para este experimento de demonstração, o tecido alvo é tecido cerebral de ratos todo. Ap homogeneizao, as amostras podem ser tratadas com detergente suave, tal como 2% de digitonina para solubilizar e permitir a electroforese de proteínas de membrana. </li><li> Transferir o homogeneizado para um tubo de microcentrífuga de 1,5 ml, e centrifugar a 14000 xg durante 30 min para sedimentar os conteúdos celulares insolúveis. after centrifugação, decanta-se o sobrenadante para um tubo limpo em gelo. </li><li> Seguir as instruções do fabricante para medir a concentração de proteína sobrenadante utilizando ácido bicinchonínico (BCA) ou um ensaio de quantificação de proteína semelhante. </li><li> Se a amostra (s) de homogenado conter detergente, adicionar uma quantidade de 5% de Coomassie Blue G-250 em solução aquosa suficiente para levar a solução homogeneizado a 0,25% de Coomassie. </li></ol> 2. BN-PAGE <ol><li> Dilui-se 25 ml de 20x azul nativa (BN) electroforese estoque de tampão (1,0 M de Bis-Tris, 1,0 M de tricina, pH 6,8) em 475 mL de dH2O contendo 0,002% de Azul de Coomassie para fazer 500 ml de tamp de corrida 1x. <ol><li> Se as amostras contêm detergente, em vez disso fazer 250 ml de tamp de corrida 1x contendo 0,02% de Coomassie e mais 250 mL contendo nenhum. </li></ol></li><li> tampão (s) frio a 4 ° C. </li><li> Limpar e montar um gel de poliacrilamida que derrama Cassete de acordo com as instruções do fabricante. </li><li&gt; Despeje o gel de poliacrilamida BN (3% de camada de empilhamento em T, 6% de camada de resolução T) de acordo com uma receita padrão 7 e coloque o pente de poço. </li><li> Após a polimerização dos géis, enxágüe a cassete com dH2O e monte o aparelho de eletroforese de acordo com as instruções do fabricante. </li><li> Remova o pente do poço e encha os poços com 1x buffer de execução BN-PAGE. Evite encher toda a câmara interna com tampão até as amostras serem carregadas. <ol><li> Se as amostras contiverem detergente, encher os poços com o tampão contendo 0,02% de Azul de Coomassie. NOTA: Execute os passos 2.7-2.9 a 4 ° C. </li></ol></li><li> Usando pontas de carga de gel, pipetar um volume de homogeneizado contendo 20 μg de proteína nos poços desejados. Pipetar um volume equivalente de 1x tampão de amostra BN-PAGE em poços não utilizados. NOTA: Utilizar a concentração de proteína determinada a partir do ensaio de BCA para calcular o volume apropriado de homogeneizado a adicionar aos poços. </li><li> Depois as amostras são carregadas, encher a câmara interior com tampão 1x BN-PAGE em execução. Certifique-se o gel é inteiramente submerso em tampão. Em seguida, encher a câmara exterior com 1x tampão de corrida para o nível indicado pelo fabricante. <ol><li> Se as amostras contêm detergente, encher a câmara interior com o tampão 1x contendo 0,02% de azul de Coomassie, e a câmara exterior com tampão de corrida 1x isento de corante. </li></ol></li><li> Ligar os eléctrodos para a fonte de alimentação, e electroforese as proteínas no gel a 150 V até que a banda de corante progride ~ 2 centímetros em resolver camada. Parar e desligar a alimentação eléctrica. </li></ol> 3. A reticulação NOTA: Efectuar os Passos 3,1-3,6 a 4 ° C. <ol><li> Desmontar o aparelho de electroforese, e separar as vidraças da cassete de gel. Remover e descartar a camada de empilhamento. </li><li> Corte cuidadosamente o gel apenas abaixo da aresta inferior da frente do corante. Levaro cuidado de fazer este corte tão lisa e direita quanto possível, e, em seguida, descartar a peça não utilizada do gel. Isto deixará uma ~ 2 cm de largura, tira horizontal em gel de poliacrilamida, que contêm toda a proteína na amostra de homogenato. </li><li> Aparar quaisquer porções não utilizadas ao longo das bordas da tira de gel. </li><li> Cuidadosamente colocar a tira em 10 mL de tampão fosfato salino (PBS), em um recipiente pequeno, e misturar suavemente por nutação durante 30 minutos para equilibrar. </li><li> Após equilíbrio, descartar e substituir o PBS com mais 10 ml. Pipetar 500 L de 25 mM de DSP dissolvido em sulfóxido de dimetilo na PBS, e continuar a misturar como anteriormente (passo 3.4), durante 30 min. </li><li> Decantar a solução de DSP. Adicionar 10 ml de 0,375 M de tris (hidroximetil) aminometano (Tris-HCl), pH 8,8, contendo 2% de dodecilsulf ato de sódio (SDS) para extinguir o DSP que não reagiu. Continuar nutação durante 15 minutos. </li></ol> 4. SDS-PAGE <ol><li> Enquanto tira de gel é têmpera, preparar SDS-PASoluções de gel GE de acordo com métodos padrão 9. Não adicione reagentes de polimerização. </li><li> Após terminar a reacção, voltar a tira de gel ΒΝ até à temperatura ambiente, e fundido a tira dentro de uma nova cassete de gel. <ol><li> Para fazer isso, cuidadosamente pegar o gel e colocá-lo sobre uma placa de gel de espaçador cassete limpo. </li><li> Oriente a tira de modo que a parte inferior da frente do corante é mais próximo à parte superior da nova cassete (ou seja, o lado que viajou mais distante durante BN-PAGE deve ser o mais próximo do topo da nova cassete, a tira de gel deve ser invertida a partir do seu prévio orientação). Ver Figura 3. </li><li> Colocar a tira de tal modo que o seu bordo superior encontra-se ainda com que a borda superior da placa de cobertura será (ou seja, ele vai estar no topo do gel de novo). Certifique-se a frente de corante é paralela às bordas horizontais da placa de vidro. </li><li> Empurrar um lado da tira excisado contra uma das paredes do espaçador, deixando espaço no tele outro lado para o gel para ser vertida e um padrão de proteína, ou escada para ser carregado. </li><li> Se a borda inferior da tira de gel contém quaisquer áreas serrilhadas ou irregulares, cortá-los cuidadosamente distância. Se estiver presente, eles vão bolhas armadilha durante gel de vazamento. </li><li> Uma vez que a tira de gel está posicionada correctamente, colocar a placa de cobertura sobre a placa do espaçador. Aplique uma leve pressão para empurrar as bolhas de ar aprisionadas. </li><li> Continuar a montar o aparelho que derrama-gel de acordo com as instruções do fabricante. </li></ol></li><li> Adicionar reagentes de polimerização para o tampão de gel de resolução, e despeje-a cassete de gel preparada, usando uma pipeta serológica. Encher a cassete de gel a aproximadamente 2 cm abaixo da tira de gel BN-PAGE excisado, para deixar espaço para a camada de empilhamento. </li><li> Adicionar 100 mL de butanol sobre a parte superior do gel derramado, e permitir 30 min para a polimerização da camada de resolver. Decantar o butanol. </li><li> Adicionar reagentes de polimerização para a solução de gel de empilhamento. Usando um serologpipeta ical, verter a camada de empilhamento para preencher todo o espaço vazio que permanece na cassete de gel. <ol><li> Inclinar a cassete de gel, como a camada de empilhamento é derramado de modo a preencher o espaço abaixo da tira de gel, e as bolhas de ar não estão presos. </li><li> À medida que o tampão de gel de empilhamento preenche o espaço vazio por baixo da tira de gel, retornar gradualmente a cassete de gel a base de nível. </li><li> Continuar a preencher o espaço vazio ao lado da tira de gel excisado com o tampão do gel de empilhamento, até que quase transborda. </li></ol></li><li> Permitir que a camada de empilhamento para polimerizar durante 30 minutos. NOTA: Adicione 10x SDS-PAGE O tampão de corrida (Tris 250 mM (hidroximetil) aminometano (tris), 1.9 M de glicina, 1% de SDS), enquanto que o gel é a polimerização. Isso também pode ser feito com antecedência e armazenado à temperatura ambiente. </li><li> Dilui-se 50 mL de 10X de SDS-PAGE estoque tampão de corrida em 450 mL de dH2O para fazer tampão de trabalho 1x. </li><li> Depois da camada de empilhamento tem polimerizado, remover a cassete de gel da vertendoaparelho, lavar com dH 2 O, e montar o aparelho de electroforese de acordo com as instruções do fabricante. </li><li> Encher a câmara interior completamente com tampão de corrida SDS-PAGE 1x, em seguida, encher a câmara exterior para o nível indicado pelo fabricante. </li><li> Carregar o espaço ao lado da tira de gel excisada com uma escada de peso molecular ou padrão de proteína apropriada. </li><li> Anexar os eléctrodos para a fonte de alimentação, e electroforese das amostras a 120 V. Quando o corante de Coomassie corre fora do gel, parar e desligar a fonte de alimentação. </li><li> Analisar o gel, utilizando métodos padrão de eiectromancheamento e detecção da proteína através da ligação 10 anticorpo. </li></ol>

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interações proteína-proteína são importantes para cada tarefa seres vivos realizar. Devido a isso, eles são objecto de intenso escrutínio e pesquisa. Multímero-PAGE é um novo método para a captura, separar, e a análise de uma grande variedade de complexos de proteínas. Foi anteriormente demonstrado a sua aplicabilidade ao estudo oligimerization da doença associada à proteína α-sinucleína 11. No entanto, é extensível para muitos complexos de proteína, tal como demonstrado na <s...

<html> Função celular normal é dependente de interacções proteína-proteína de 1, 2. Como resultado, as doenças humanas são frequentemente caracterizadas por perturbações na montagem e comportamento de vários complexos de proteínas 3. A capacidade de caracterizar essas interacções é, por conseguinte, crítica. meios actuais de detecção destas interacções requerem purificação de proteínas alvo, muitas vezes seguidos por suspenso de seus parceiros interactuantes. Clássica de purificação é realizado por centrifugação diferencial, precipitação e / ou cromatograf ia em 4. Estes métodos são demorados, deve ser alterado para cada proteína alvo, e muitas vezes resultam em baixos rendimentos. Modernos métodos de purificação envolvem a fusão de sinais peptídicos a proteínas alvo, seguido de imunoprecipitação ou de extracção em colunas carregadas com esferas ligadas a uma molula de captura 5,&quot;&gt; 6. Embora esta seja extensível para muitas proteínas, que exige modificação da sequência do alvo, potencialmente resultando em construções que diferem na afinidade para os seus parceiros de ligação usuais. A natureza delicada de algumas interacções proteína-proteína significa que este método pode não ser aplicável para muitos cenários. Além disso, os ensaios de pull-down utilizadas para mapear as interações proteína-proteína pode não capturar uma imagem celular precisa, devido à graus restritos de liberdade e os níveis de não-nativos da proteína isca. Idealmente, os complexos de proteína podia ser detectada nos seus estados nativos, sem a necessidade de purificação ou suspenso. Electroforese em gel de poliacrilamida nativa azul (BN-PAGE) foi desenvolvido como uma alternativa menos desnaturante a electroforese em gel de poliacrilamida dodecil sulfato de sódio (SDS-PAGE), e permite a separação de algumas proteínas e complexos a partir de amostras biológicas 7. No entanto, as proteínas em BN-PAGE separadas com base em um large número de variáveis, incluindo tamanho, carga, estrutura tridimensional, e associação com outras moléculas. As interacções destes factores resultam frequentemente em co-separação de proteínas, formação de agregados, e pobre resolução banda de proteína. Electroforese em gel nativo de poliacrilamida bidimensional resolve alguns, mas não todos, estes problemas 8. Para contornar as complicações associadas com a separação nativa, alguns autores utilizam reagentes reactivos de amina de reticulao, tais como ditiobis (propionato de succinimidilo) (DSP), para capturar os complexos de proteína em lisados de tecido 4. Estes lisados ​​tratados pode então ser desnaturado e separadas por SDS-PAGE, preservando ao mesmo tempo o tamanho nativo e a composição de complexos de proteínas. No entanto, uma vez que os reagentes de reticulação reagir com base na proximidade de uma molécula para outra, e proteínas em lisados ​​de tecido tem muitos graus de liberdade e pode interagir estocasticamente, inespecífica transversal fundo-linking pode ser elevado, especialmente em amostras concentradas. Isso pode levar a resultados difíceis de interpretar. Aqui, demonstramos a utilização de um método híbrido BN-PAGE / SDS-PAGE, denominada electroforese em gel de poliacrilamida-multero (multímero-PAGE), para separar e detectar os conjuntos de proteínas em misturas complexas. Inicialmente, lisado de células é suspenso em gel de poliacrilamida através BN-PAGE. O gel contendo o lisado é então feito reagir com o reagente DSP reticulação. e ligeiramente separadas no gel imobilizada-pseudo, as proteínas são muito menos susceptíveis de reagir de forma não específica, o que significa que a reactividade cruzada de fundo ligante é reduzida. Após a reticulação, as bandas de gel são excisadas e separados por meio de SDS-PAGE. O gel resultante pode então ser analisada por quaisquer meios tipicamente associados com electroforese em gel de poliacrilamida. Este método permite a separação e a detecção de complexos de proteínas nativas em lisado de tecido não modificada, sem a necessidade de purificação adicional ou puxar-Down.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;epsilon;-Aminocaproic acid</td><td>Sigma</td><td>A2504</td><td></td></tr><tr><td>Acrylamide</td><td>Acros Organics</td><td>164855000</td><td>Toxic.</td></tr><tr><td>Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td><td>BioRad</td><td>161-0148</td><td>Toxic.</td></tr><tr><td>Ammonium persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>Anti rabbit IgG-HRP from goat</td><td>Santa Cruz Biotechnology</td><td>sc-2004</td><td></td></tr><tr><td>Bicinchoninic acid assay kit</td><td>Thermofisher</td><td>23225</td><td></td></tr><tr><td>Bis-tris</td><td>Sigma</td><td>B9754</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Butanol</td><td>Fisher Scientific</td><td>A399-1</td><td></td></tr><tr><td>Chemiluminescence substrate kit</td><td>ThermoFisher</td><td>24078</td><td></td></tr><tr><td>Coomassie blue G-250</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Toxic.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>D128-1</td><td></td></tr><tr><td>Dithiobis(succinimidylpropionate)</td><td>Thermo Scientific</td><td>22585</td><td></td></tr><tr><td>Dry nonfat milk</td><td>LabScientific</td><td>M0841</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>Thermofisher</td><td>78430</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144SI-212</td><td>For titration. Caustic.</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-4</td><td>For PVDF membrane activation. Toxic.</td></tr><tr><td>Monoclonal anti &amp;alpha;-synuclein IgG from rabbit</td><td>Santa Cruz Biotechnology</td><td>sc-7011-R</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-tetramethylethylenediamine</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>N,N&#039;-methylenebisacrylamide</td><td>Acros Organics</td><td>16479</td><td>Toxic.</td></tr><tr><td>NP40</td><td>Boston Bioproducts</td><td>P-872</td><td></td></tr><tr><td>Polysorbate 20 (tween-20)</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Polyvinylidene fluoride transfer membranes</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217-3</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Thermo Scientific</td><td>88265</td><td></td></tr><tr><td>SDS solution (10% w/v)</td><td>BioRad</td><td>161-0416</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma</td><td>L37771</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Fisher Scientific</td><td>BP329-1</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-500</td><td></td></tr><tr><td>Tris-HCl (0.5 M buffer, pH 6.8)</td><td>BioRad</td><td>161-0799</td><td></td></tr><tr><td>Tris-HCl (1.5 M buffer, pH 8.8)</td><td>BioRad</td><td>161-0798</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE Imagequant LAS 4000</td><td>GE Healthcare</td><td>28-9558-10</td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td></td></tr><tr><td>Synergy H1 microplate reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Gel Former + Stand</td><td>Biorad</td><td></td><td></td></tr><tr><td>Microfuge 22R centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>2 mL dounce homogenizer</td><td></td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Ultrasonic tissue homogenizer</td><td>Fisher Scientific</td><td>FB120220</td><td></td></tr></tbody></table>

multimer-page: a method for capturing and resolving protein complexes in biological samples

Existem muitos métodos bem desenvolvidos para purificar e estudar proteínas individuais e peptídeos. No entanto, a maioria das funções celulares são realizados por redes de complexos de proteínas que interagem, que são muitas vezes difíceis de investigar, porque a sua ligação é não-covalente e facilmente perturbado por técnicas de purificação. Este trabalho descreve um método de estabilização e separando complexos de proteína nativa a partir de tecido não modificado utilizando electroforese em gel de poliacrilamida bidimensional. lisado de tecido é carregado num gel de poliacrilamida nativo azul-n desnaturante, em seguida, uma corrente eléctrica é aplicada até que a proteína migra uma curta distância dentro do gel. A tira de gel contendo a proteína migrado é então excisada e incubadas com as ditiobis reactivos de amina reagente de ligação cruzada (propionato de succinimidilo), que estabiliza covalentemente complexos de proteínas. A tira de gel contendo complexos de ligação cruzada é então moldada num gel de poliacrilamida de sulfato de dodecilo de sódio, e tele complexos são separados completamente. O método baseia-se em técnicas e materiais familiares para a maioria dos biólogos moleculares, o que significa que é barato e fácil de aprender. Enquanto que é limitada na sua capacidade para separar adequadamente extremamente grandes complexos, e não tem sido universalmente bem sucedida, o método foi capaz de capturar uma grande variedade de complexos bem estudadas, e é provavelmente aplicável a muitos sistemas de interesse.

Neste experimento de demonstração, multero-PAGE foi realizada em toda lisado cérebro de rato. As proteínas resultantes separadas foram transferidas para membranas de difluoreto de polivinilideno (PVDF), e em seguida sondadas com anticorpos contra proteínas que são conhecidas por formar complexos. A Figura 1 mostra uma validação do protocolo por dois meios. Em primeiro lugar, demonstra-se que as proteínas de ligação cruzada são susceptíveis ao corte por adiç...

Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

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

Um método para estabilizar e separando complexos de proteína nativa a partir de lisado de tecido não modificado utilizando uma proteína de reticulação reactivo com amina ligado a uma electroforese em gel de poliacrilamida bidimensional novo sistema (PAGE) é apresentada.

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 ...

multimer-page-method-for-capturing-resolving-protein-complexes

Research

JoVE Journal

Biochemistry

11.3K visualizações.  Michigan State University.  Um método para estabilizar e separando complexos de proteína nativa a partir de lisado de tecido não modificado utilizando uma proteína de reticulação reactivo com amina ligado a uma electroforese em gel de poliacrilamida bidimensional novo sistema (PAGE) é apresentada. 

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

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

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 ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

Analisando a dinâmica da proteína montada sobre e libertado da interferometria Biolayer Biosensor usando espectrometria de massa e de microscopia eletrônica

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Perfilamento Complexome de alta resolução por Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Fluxo de trabalho de espectrometria de massa de imunoprecipitação sem rótulos para perfis de interactom nuclear em larga escala

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

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

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

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

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

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 ...

Biologia

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Analisar grandes complexos de proteínas por espectrometria de massa estruturais

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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 ...

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 ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Descobrindo Interações proteína e caracterização da função das proteínas Usando HaloTag Tecnologia

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...