Os autores agradecer os Drs Jim Lah e Allan Levey, Emory departamento de Neurologia, sugestões e comentários úteis. Este trabalho foi parcialmente financiado pela parceria de medicina acelerando subvenção (U01AG046161-02), o Emory Alzheimer centro de pesquisa da doença (P50AG025688) e um Instituto Nacional sobre envelhecimento concedem (R01AG053960-01) para N.T.S. Esta pesquisa também foi apoiada em parte pelo núcleo neuropatologia da subvenção Emory neurociência NINDS núcleo instalações, P30NS055077.

declaração de ética: todos os tecidos do cérebro foram obtidos a partir da Emory Alzheimer &#39; s doença Research Center (ADRC) banco de cérebro. Tecidos humanos após a morte foram adquiridos ao abrigo de protocolos apropriados institucional Review Board (IRB).
 1. homogeneização e fracionamento <ol><li>seleção de tecido Nota: congelado o tecido do córtex frontal após a morte de controle saudável (Ctl) e patologicamente casos confirmados de AD foram seleccionados a partir do Emory Banco de cérebro ADRC (n = 2). Post-mortem neuropatológica avaliação da distribuição de placa amiloide foi realizada de acordo com o consórcio para estabelecer um registro para Alzheimer &#39; s doença semi-quantitativa marcando critérios 24, embora patologia do tangles emaranhado foi avaliada em conformidade com o sistema de preparo de Braak. 16 <ol><li>obter congelado o tecido cerebral post-mortem de controle saudável (Ctl) e patologicamente confirmado casos de AD. Utilizando recipientes de gelo seco, fórceps e uma lâmina de barbear, impostos especiais de consumo ~ porções de 250 mg de matéria cinzenta de cada amostra de tecido na tarada descartável pesam barcos, tendo o cuidado de evitar que o tecido descongelar. Registrar o peso de cada peça para ser homogeneizado.</li>
		<li>Impostos ~ 250 mg de matéria cinzenta, usando a pinça e navalha, inspecionando visualmente para localizar a interface entre a substância cinzenta externa e interna de substância branca. Evitar a substância branca e, mais importante, qualquer grandes vasos sanguíneos ou regiões sangrentas, meninges ou aracnoide. Mantenha o tecido congelado durante o processo de corte a trabalhar rapidamente e frequentemente colocando o barco pesagem com tecido cerebral em recipientes de isopor com gelo seco</li>
		<li>Gravar o peso exato da matéria cinzenta extirpado da cada pedaço congelado usando uma balança analítica. Calcular o volume de tampão baixo sal (LS) (ver tabela 1) necessário para homogeneização de homogenizacao (5 mL/g ou 20% w/v).</li>
		<li>Preparar 10 mL LS de tampão com 1x do inibidor de protease e fosfatase cocktail e calma na Ice.</li>
		<li>Remover tecido pesado e pesagem do barco de gelo seco e corte em pedaços de 2 x 2 mm aproximadamente, enquanto ele derrete. Transferência para um tubo de homogeneizador de pre-refrigerados homogenizacao 2ml no gelo</li>
		<li>Adicionar 5 mL/g (20% w/v) do buffer de sal baixo gelada (LS) com 1 X coquetel de inibidor de protease e fosfatase.</li>
		<li>Homogenize no tecido cerebral no gelo usando aproximadamente 10 traços de alta apuramento pilão A e 15 golpes de pilão de depuração baixa B.</li>
		<li>Transferir tudo homogeneizado para um tubo de polipropileno 2 mL, usando um pipeta Pasteur de vidro. Rótulo do tubo com &#8220; TH &#8221; (Homogenate Total), o tecido caso número e homogeneizado volume. alíquota 0,8 mL num tubo rotulado 1,5 mL. Qualquer excesso homogenate pode ser similarmente aliquotadas e armazenado a -80 ° C.</li>
		<li>a cada 0,8 mL alíquota, adicionar 100 µ l cada de 5 M NaCl e sarkosyl de 10% (p/v) para as concentrações de 0,5 M e 1% p/v, respectivamente. Misturar os tubos bem pela inversão e incubar no gelo por 15 min. de tubos de rótulo com &#34; TH-S &#34; (Total homogeneizado-Sarkosyl) para indicar que o homogenates estão no buffer de sarkosyl (tabela 1).</li>
		<li>Proceda à sonicação cada tubo para pulsos de três 5 s em 30% amplitude no gelo (intensidade máxima = 40%) usando uma sonda de microtip.</li> <li>determinar as concentrações de proteína do homogenates usando o ácido bicinchoninic (BCA) ensaio 25 método. 
		Nota: A concentração de proteína média de TH-S homogenates preparado em 5 mL LS por grama de tecido é de 15-20 mg/mL. O homogenates pode ser fracionada imediatamente ou armazenado a-80 ° C até o uso.</li>
		<li>Homogenates TH-S diluir a 10 mg/mL, usando gelada sark buffer (tabela 1) com 1 x inibidores de protease e fosfatase.</li>
		<li>Transferir 5 mg proteína (0,5 mL) de cada homogenate TH-S em 500 tubos de se µ l em policarbonato e par-equilíbrio com buffer de sark. Carregar os tubos em um rotor pre-refrigerado e se a 180.000 x g durante 30 min a 4 ° C. transferência do sarkosyl-solúvel fracções sobrenadantes (S1) para tubos de 1,5 mL e armazene a -80 ° C. 
		Nota: (Lavagem opcional) adicionar 200 µ l de sark-buffer para o se tubos contendo as frações insolúveis em detergente (P1) e desalojar as pelotas (P1) da parte inferior dos tubos se utilize uma ponta de pipeta de 200 µ l.</li>
		Tubos <li>brevemente de pulso-gire o invertido para 2-3 s (&#8804; 2.500 x g) em um microcentrifuge para transferir o buffer e pelotas (P1) para os tubos de 1,5 mL abaixo. Resuspenda as pelotas insolúveis (P1) no buffer de sark pipetando para cima e para baixo para garantir a pelota é interrompida.</li>
		<li>Par-equilíbrio e centrifugar 180.000 x g por um adicional 30 min a 4 ° C.</li>
		<li>Desprezar o sobrenadante de lavagem opcional (S2) e incube as pelotas de sarkosyl insolúveis (P2) em 50-75 µ l de tampão de ureia (tabela 1) com 1 x PIC (protease e fosfatase inibidor cocktail) por 30 min à temperatura ambiente para solubilizar o pelota. 
		Nota: Buffer de ureia quente a temperatura ambiente antes do uso para evitar a precipitação de SDS. Para aplicações sensíveis ao detergente, omitir SDS de buffer de ureia e considerar lavar o sedimento insolúvel (P2) no baixo sal buffer antes de resuspending no buffer de ureia.</li>
		<li>Transferir as ressuspensa Pelotas (P2) para tubos de 0,5 mL e usar breve (1 s) microtip sonication em amplitude de 20% (intensidade máxima = 40%) para solubilizar totalmente as pelotas.</li>
		<li>Determinar as concentrações de proteína de sarkosyl-solúvel (S1) e insolúveis (P2) frações usando o BCA método de ensaio. Use estas frações imediatamente ou armazenar a-80 ° C até o uso.</li>
	</ol></li></ol> 2. Immunoblotting <ol><li>mancha ocidental Nota: mancha ocidental foi realizada de acordo com os procedimentos anteriormente relatados com ligeiras alterações. 5 , 10 <ol><li>preparar 40 µ g amostras de homogenates total (TH-S), sark-solúvel (S1) e frações de sark insolúveis (P2) em 1 amortecedor da amostra X Laemmli SDS-page com pH TCEP 5 mM 7. Incubar as amostras a 95 ° C por 5 min.</li>
		Amostras de <li>carga em SDS-PAGE um pré-moldado bem 10 4-12% Bis-Tris gel. Electrophorese em 80 V para o primeiro centímetro (10 min) e 120 V para o restante (60 min), ou até que a tintura de seguimento atinge a parte inferior do gel.</li>
		<li>Use uma lâmina de barbear fresca e gel de faca para dividir-abrir a gaveta do gel de pre-cast. Delicadamente cortar fora os pentes e inferior do gel com uma lâmina de barbear fresco.</li>
		<li>Resuspenda as pelotas insolúveis (P1) em 200 µ l sark-buffer com 1x PIC pipetando cima e para baixo.</li>
		<li>Transferência para uma membrana de nitrocelulose de 0,2 µm usando um método de transferência de seco em uma máquina de mancha (consulte a tabela de materiais).</li>
		<li>Bloquear a membrana em tampão de bloqueio (BB) sem Tween 20 por 30 min, seguida com BB com 0,05% Tween 20 (BB/T) por 30 min. Após o bloqueio, lavar a membrana em TBS/T (0.1% Tween 20) por 5 min remover o tampão de bloqueio excesso.</li>
		<li>Preparar diluições de 1:1,000 de pT231 os anticorpos primários, Tau-2 (pan tau), AT8 e EEA1 no BB/T (0,05% Tween 20).</li>
		<li>Incubar as membranas em solução de anticorpo primário durante a noite a 4 ° C, com agitação circular. Lavar a membrana em TBS/T 3 x 15 min.</li>
		<li>Sonda a membrana com uma diluição de 1: 20.000 de fluorescente-etiquetadas infravermelho próximo anticorpo secundário no BB/T para 60 min à temperatura ambiente na escuridão agitador. Lavar a membrana para 3 x 10 min na TBS/T e 2 x 5 min em TBS</li>
		<li>Scan a membrana usando um sistema no comprimento de onda de excitação apropriada (por exemplo, 700 nm (canal vermelho) para tintura infravermelho 680 cabra anti-rato IgG (H + L) secundário e 800 nm (canal verde) para tintura infravermelho 790 burro anti-rabbit IgG (H + L de imagens de infravermelho secundária)).</li>
		<li>Usar o software de imagem para quantificar a intensidade do sinal e executar medidas de densitometria conforme o fabricante &#39; instruções de s, garantir autoconfiguração de fundo é definido como média o fundo todo.</li>
	</ol>
	</li>
</ol>

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Neste documento vamos apresentar e discutir um protocolo de detergente-fracionamento de etapa única Abreviado que é aplicável a uma ampla variedade de aplicações experimentais que variam de análise proteômica de espectrometria de massa-baseado a proteína funcional ensaios de enrolamento e mancha ocidental. 5 , 6 , 7 , 10 esta metodologia é talvez mais eficaz quando usado para estudar p...

Doenças neurodegenerativas, como a doença de Alzheimer (AD), a doença de Parkinson (PD), a doença de Huntington (HD), esclerose lateral amiotrófica (ela) e as doenças de príon estreitamente relacionadas são proteinopathies caracterizadas pelo acúmulo gradual de agregados de proteína insolúvel em detergente no cérebro com acompanhamento cognitivo declinam. 1 , 2 este recurso compartilhado patológico é pensado para jogar um papel central na etiologia e fisiopatologia dessas doenças neurodegenerativas. 2 estes agregados, normalmente, compostos de fibras de amiloides poliméricas, que são compostas de repetir unidades de misfolded proteínas exibindo Cruz β-estrutura. 1 , 2 , 3 , 4 bioquimicamente, agregados amiloides são altamente resistentes à desnaturação química ou térmica e solubilização,3 que apresenta desafios únicos para a sua purificação, análise e estudo através de técnicas bioquímicas tradicionais. 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 não é novidade, a fração de proteína insolúvel em detergente tem sido o foco de muita pesquisa em fisiopatologia das doenças neurodegenerativas que envolvem o acúmulo de misfolded proteínas. 6 , 12 , 13 , 14
Técnicas de fracionamento bioquímico frequentemente têm sido utilizadas para enriquecer a fração insolúvel em detergente de homogenates cerebral post-mortem. 6 , 12 , 13 , 14 um dos métodos mais comuns envolve a extração sequencial de tecido homogenates com buffers e detergentes de aumentar o rigor, seguido por ultracentrifugação para particionar as frações solúveis e insolúveis. Um protocolo comumente usado fracionamento sequencial6,14 envolve a homogeneização de amostras de tecido congelado em um buffer de detergente livre baixo sal (LS) e as pelotas insolúveis resultantes então são extraídas sequencialmente com amortecedores que contêm sal elevado, detergentes não-iônicos, alta sacarose, detergentes iônicos e finalmente chaotropes como ureia. 6 , 14 uma desvantagem óbvia de tais protocolos um fracionamento sequencial é o tempo substancial e compromisso de trabalho necessário para concluí-las. Incluindo a homogeneização e ultracentrifugação, um protocolo típico de cinco etapas de fracionamento pode levar várias horas ou mesmo dias para completar. 4 , 6 , 7 , 10 , 15 , 16 , 17 , 18 além disso, como muitos agregados de proteína patológica permanecem insolúveis em todos, mas as mais adversas condições19,20 a maioria das frações geradas são de valor limitado. Assim, as etapas de fracionamento menos rígidas utilizando altas concentrações de sal e detergentes não-iônicos são em grande parte redundantes.
Estudos anteriores mostraram que o detergente iônico N-Lauril-sarcosina (sarkosyl) é um excelente candidato para um protocolo simplificado fracionamento de detergente de etapa única. 5 , 6 , 12 , 13 , 14 , 21 , 22 , 23 como um detergente de desnaturação, sarkosyl é suficientemente rigorosa para solubilizar a maioria vasta das proteínas nativamente dobradas no cérebro sem solubilizing agregados de misfolded proteínas compostos por beta-amiloide (Aβ),6,11 tau fosforilada (pTau),6 TAR ADN-proteína obrigatória 43 (TDP-43),14 alfa-synuclein,12,13 do tremor epizoótico,23 ou U1 pequenas ribonucleoproteínas nucleares (snRNPs U1) como U1 - 70 K. 5 , 21 , 22 Como sarkosyl é menos rigoroso do sulfato dodecyl de sódio detergente aniônico onipresente (SDS), preserva menos robustas oligoméricas formas de agregados de misfolded proteínas que não podem suportar o tratamento de SDS. 9
Anteriormente, nós descrevemos um protocolo de detergente-fracionamento Abreviado que obtiveram resultados comparáveis aos métodos de fracionamento sequencial mais trabalhosos. 5 , omitindo as menos rigorosas etapas de fracionamento, fomos capazes de desenvolver um protocolo facile fracionamento de etapa única para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte. 5 este protocolo detalhado aqui descrito é bem adequado para uma ampla gama de aplicações experimentais que variam de mancha ocidental e ensaios de espectrometria de massa-baseado proteomics para enrolamento de proteína funcional e propagação de agregação. 5 , 6 , 21

<table>
	<tbody>
		<tr>
			<td>Protease and phosphatase inhibitor cocktail, EDTA-free (100X)</td>
			<td>Thermo Fisher</td>
			<td>78441</td>
			<td>protease &#38; phosphatase inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator System (ultrasonicator)</td>
			<td>Fisher Scientific</td>
			<td>FB505110</td>
			<td>microtip ultrasonicator</td>
		</tr>
		<tr>
			<td>Optimax TLX Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>361545</td>
			<td>refrigerated ultracentrifuge</td>
		</tr>
		<tr>
			<td>TLA120.1 rotor</td>
			<td>Beckman Coulter</td>
			<td>362224</td>
			<td>ultracentrifuge rotor</td>
		</tr>
		<tr>
			<td>500 ul (8x34mm) polycarbonate tubes, thickwall</td>
			<td>Beckman Coulter</td>
			<td>343776</td>
			<td>ultracentrifuge tubes for TLA120.1 rotor</td>
		</tr>
		<tr>
			<td>4X SDS sample buffer</td>
			<td>Home-made</td>
			<td>N/A</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>TCEP solution, neutral pH</td>
			<td>Thermo Fisher</td>
			<td>77720</td>
			<td>reducing agent</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer</td>
			<td>Thermo Fisher</td>
			<td>37542</td>
			<td>blocking buffer</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer + 0.05% Tween 20</td>
			<td>Thermo Fisher</td>
			<td>37543</td>
			<td>blocking buffer and antibody diluent</td>
		</tr>
		<tr>
			<td>4-12% Bolt Bis-Tris Plus gels, 10-well</td>
			<td>Thermo Fisher</td>
			<td>NW04120BOX</td>
			<td>precast SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>MES SDS Running Buffer (20X)</td>
			<td>Thermo Fisher</td>
			<td>B0002</td>
			<td>SDS-PAGE running buffer</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt (sarkosyl)</td>
			<td>Sigma Aldrich</td>
			<td>L5777-50G</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>1.5 ml polypropylene Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749521-1500</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>cordless motor for Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749540-0000</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>Anti-Tau-2 (pan tau) antibody</td>
			<td>Chemicon</td>
			<td>MAB375</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-threonine 231 Tau antibody</td>
			<td>Millipore</td>
			<td>MAB5450</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)</td>
			<td>Thermo Fisher</td>
			<td>MN1020</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-early endosome antigen 1 (EEA1) antibody</td>
			<td>abcam</td>
			<td>ab2900</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A21058</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A11374</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>iBlot2 Dry Blotting System</td>
			<td>Thermo Fisher</td>
			<td>IB21001</td>
			<td>Gel transfer</td>
		</tr>
		<tr>
			<td>iBlot2 Transfer Stacks, Nitrocellulose, mini</td>
			<td>Thermo Fisher</td>
			<td>IB23002</td>
			<td>Gel transfer</td>
		</tr>
	</tbody>
</table>

enrichment of detergent-insoluble protein aggregates from human postmortem brain

Neste estudo, descrevemos um protocolo abreviado fracionamento de etapa única para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte. O detergente iônico N-Lauril-sarcosina (sarkosyl) efetivamente solubiliza proteínas dobradas nativamente no tecido cerebral, permitindo o enriquecimento de agregados da proteína insolúvel em detergente de uma ampla gama de proteinopathies neurodegenerativas, tais como A doença de Alzheimer (AD), doença de Parkinson e esclerose lateral amiotrófica e doenças de príon. Controle humano e tecidos do cérebro após a morte de AD foram homogeneizadas e sedimentada por ultracentrifugação na presença de sarkosyl para enriquecer agregados de proteína insolúvel em detergente incluindo tau fosforilada patológica, o componente do núcleo do tangles emaranhados em AD. Mancha ocidental demonstrou a solubilidade diferencial de agregados-tau fosforilada e a proteína solúvel em detergente, Early Endosome antígeno 1 (EEA1) no controle e no cérebro do AD. Análise proteômica também revelou o enriquecimento de β-amiloide (Aβ), tau, snRNP70 (U1 - 70 K) e apolipoproteína E (APOE) nas fracções sarkosyl insolúveis do cérebro AD comparados de controle, consistente com estratégias de fracionamento de tecido anterior . Assim, o presente protocolo simples enriquecimento é ideal para uma ampla gama de aplicações experimentais variando de mancha ocidental e proteína funcional ensaios de agregação co a espectrometria de massa-baseado proteomics.

O protocolo de sarkosyl de etapa única abreviado-fracionamento foi usado para enriquecer agregados de proteína insolúvel em detergente de controle e o cérebro após a morte de AD (Figura 1). Proteínas de frações TH-S, S1, S2 e P2 foram resolvidas por SDS-PAGE, fixado por 15 min em Coomassie azul reserva fixador seguida de coloração suave com buffer de coloração azul brilhante de Coomassie G-250. A etapa de ressuspensão é opcional, desde que não ...

Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

Um protocolo de fracionamento abreviado para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte é descrito.

Enriquecimento de agregados da proteína insolúvel em detergente do cérebro humano após a morte

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

enrichment-detergent-insoluble-protein-aggregates-from-human

Research

JoVE Journal

Biochemistry

12.0K visualizações.  Emory School of Medicine. Um protocolo de fracionamento abreviado para o enriquecimento dos agregados de proteína insolúvel em detergente do cérebro humano após a morte é descrito.

Vídeo: Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

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

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

A purificação de afinidade em tandem de complexos de proteínas a partir de células eucarióticas

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Digestão de proteínas, ultrafiltração e cromatografia de exclusão para otimizar o isolamento do Exosomes do Plasma de sangue humano e soro

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Livre de calibração em Vitro quantificação da proteína Homo-oligomerização usando instrumentação comercial e livre, Open Source Software de análise de brilho

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Medição de alta sensibilidade de afinidades de ligação do fator-DNA transcrição por titulação do competidor usando microscopia de fluorescência

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Análise de proteínas, transporte e degradação em células vivas por pulso radioativo Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Detecção de locais de ubiquitinação de proteínas por enriquecimento de peptídeos e espectrometria de massa

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Robusta comparação dos níveis de proteína através de tecidos e durante todo o desenvolvimento usando mancha ocidental quantitativa padronizada

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Enriquecimento de uma única etapa de um histone TAP-Tagged deacetilase do fungo filamentoso Aspergillus nidulans para o ensaio de atividade enzimática

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

Triagem de inibidores da quinase em Microarrays de proteínas humanas Automontados

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...