The authors would like to acknowledge Dr. John Sheehan and Dr. Lubna Abdullah for their guidance and mentoring that were central in the completion of this work. This work was supported by funds from the National Institutes of Health (P01HL108808, UH2HL123645) and the Cystic Fibrosis Foundation Therapeutics, Inc. (EHRE07XX0). Kathryn Ramsey is supported by an NHMRC Early Career Research fellowship.

1. Prepare tampões para mucina Gel Western Blotting <ol><li> Prepare 1 litro de tampão 50x TAE (Tris-acetato-EDTA). <ol><li> Em 700 ml de água destilada (dH2O) adicionar 242 g de Tris base (0,4 M), 57,1 g de ácido acético glacial (peso líquido) (0,2 M) e 14,61 g de ácido etilenodiaminotetracético (EDTA) (50 mM). </li><li> Ajustar o pH para 8,0 e perfazer o volume a 1 litro com dH2O </li></ol></li><li> Preparar 10 ml de 10x tampão de carga. <ol><li> Prepare 5 ml de tampão 1 x TAE. Para fazer isso, adicionar 5 ml de glicerol (50%), 25 mg de azul de bromofenol (0,25%), e 100 mg de sulfato de dodecilo de sódio (SDS) (1%). </li></ol></li><li> Prepare 2 litros de tampão 20x citrato de sódio solução salina (SSC). <ol><li> Em 1,5 litros de dH2O, adicionar 350,6 g de NaCl (3 M) e 176,4 g de citrato trissódico (Na 3 C 6 H 5 O 7) (0,3 M). Ajustar o pH para 7,0 e levar o volume a 2 litros com dH 2 O. </li></ol></li><li> Prepare 1 litro de tampão 1 x TAE-SDS a 0,1%. <ol><li> Adicionar 20 ml de 50x TAE a 980 ml ​​de dH 2 O. Adicionar 1 g de SDS para fazer uma solução de SDS-TAE a 0,1%. </li></ol></li></ol> 2. TAE-SDS Agarose Gel de Preparação <ol><li> (. Isto é, 150 ml) Pour volume suficiente de TAE 1X-SDS a 0,1% de tampão em um balão de Erlenmeyer de microondas para fazer uma 5 - gel de sete milímetros de espessura. Adicionar 0,8% (1,2 g) pó de agarose. </li><li> frasco de micro-ondas em intervalos de 30 seg com agitação intermitente, até estar completamente dissolvido de agarose (normalmente 1-3 minutos). Use almofadas de mão quentes durante este processo. Tenha cuidado com a fervura enquanto agita. </li><li> Permitir que a solução de agarose para esfriar por 5 - 10 min. Nota: A solução de agarose estará pronto para ser derramado quando fundo do balão pode ser deixado na palma da mão durante 5 seg. </li><li> Prepare a bandeja de fundição de electroforese (10 x 15 cm) por meio de selagem com as arestas da fita e colocando assim pentear em posição. </li><li> Pour solução de agarose lentamente em tele fundição bandeja para evitar a formação de bolhas. Remova as bolhas usando uma ponteira. Aguarde até que o gel para esfriar e completamente solidificar. Uma vez solidificado, remover o pente lentamente para evitar o colapso dos poços por sucção e remova a fita a partir das bordas do tabuleiro. </li><li> Coloque gel de agarose na caixa de gel e encher a unidade de electroforese com tampão% SDS 1x-0,1 TAE até que o gel é totalmente coberto. </li></ol> 3. Amostra de carga e Eletroforese Separação Nota: Em nosso laboratório, nós geralmente usam amostras de mouse e os seres humanos, incluindo fluido de lavado broncoalveolar (LBA; ambas as espécies), lavagem de células a partir de células humanas epiteliais brônquicas (HBE), e amostras de saliva e escarro humanos. As amostras podem ser desnaturada em ureia 6 M no momento da recolha ou armazenada por curtos períodos de tempo (6 horas) por adição de cocktail inibidor de proteinase. <ol><li> Preparar as amostras para carregar pela adição de 10% de corante de carregamento de cada amostra (isto é., 30 ul de amostra e trêsul de corante). Homogeneizar amostras pipetando cima e para baixo. Resumidamente girar tubos a 5000 rpm para recolher gotas. </li><li> carregar cuidadosamente igual volume de amostras nas cavidades. <ol><li> dicas pré-molhados e / ou utilizar pipetas de deslocamento positivo para facilitar o carregamento de amostras altamente viscosas. Lentamente aspirado de 80 - 90% de solução de amostra-corante (ou seja, 27-30 ul) para evitar bolhas de pipetagem. </li><li> carregar lentamente no fundo dos poços e mover progressivamente a ponta da pipeta para cima. Para as amostras que permanecem ligados à ponta da pipeta, dispensar a um ângulo de 45 ° e alongar linear acima do poço ou suavemente usar o lado do poço para quebrar o muco viscoso. Não sobrecarregue os poços. </li></ol></li><li> Ligar os eléctrodos a partir da caixa do gel para o gerador de energia electroforese. Assegurar que os eletrodos estão ligados correctamente (ie., Chumbo vermelho ligado à saída positiva do gerador) para permitir carregados negativamente mucinas a correr para o lado positivo<html> eléctrodo. </li><li> Submeter o gel a 80 volts durante 90 min para um único gel de pente, ou 60-75 minutos para um gel de pente duplo para evitar sobreposição entre as duas linhas. </li><li> Desligue a alimentação do gerador e desconectar os eletrodos da fonte de alimentação. </li><li> remover cuidadosamente o gel a partir da caixa do gel com uma mão em cada lado da bandeja de vazamento para assegurar o gel permanece no lugar. </li></ol> 4. Reduzir Agarose Gel para Transferência de mucina Eficiente <ol><li> Com cuidado, coloque o apartamento gel em um recipiente de vidro. Lavar o gel com dH2O para remover vestígios de tampão TAE-SDS. </li><li> Prepare 1 litro de tampão SSC 4x por adição de 200 ml de 20x SSC em 800 ml de dH 2 O. Adicione 200 ml de 10 mM de ditiotreitol (DTT) 4x SSC solução por adição de 309 mg de DTT fresco em 200 ml de tampão SSC 4x. </li><li> Mergulhe o gel com tampão e cobertura TDT-4x SSC. Agite suavemente durante 20 minutos (10 rotações por minuto). Lavar o gel com tampão de 4x SSC TDT-free. </li></ol>&quot;&gt; 5. Vácuo Assembleia Blotter e Transferência de exemplo a um Nitrocellulose Membrane <ol><li> Prepare mata-borrão de vácuo para a transferência. <ol><li> Cuidadosamente corte membrana de nitrocelulose em dimensões ligeiramente maior do que o gel (ie., 10 x 15 cm). </li><li> Esteira porosa molhado com dH2O e colocá-lo liso-lado-up no mata-borrão. </li><li> membrana de nitrocelulose molhado com tampão SSC 4x DTT-livre e colocá-lo no centro do tapete poroso. </li><li> Cubra a esteira porosa com a junta de borracha, deixando a abertura da junta precisamente alinhada com a membrana de nitrocelulose. Se esteira porosa é ainda visível, ajuste cuidadosamente a membrana para garantir que não haja lacuna permanece entre a junta e membrana. </li></ol></li><li> Coloque cuidadosamente o gel de agarose na parte superior da membrana. Certifique-se de formas de gel de uma vedação estanque com a junta e os poços do gel estão posicionados sobre a membrana de transferência. <ol><li> Evitar a formação de bolhas entre a membrana e o gel. Para minimizar oformação de bolhas, esguiche algumas gotas de 4x SSC tampão sobre a membrana e o gel deslizar de cima para baixo para lavar quaisquer bolhas formadas. Evitar mover o gel na membrana como proteínas começarão a blot imediatamente. </li></ol></li><li> cobrir cuidadosamente o gel com tampão de 4x SSC. </li><li> Comece a transferência de mucina por sucção. <ol><li> Vire mata-borrão de vácuo ON e pressão fixado em 40 - 50 mbar. Adicionar algumas gotas de tampão SSC 4x no gel a cada 5-10 minutos para evitar que o gel seque. transferência de correr por 90 min. Opcional: Após a conclusão, marque os poços com um lápis para orientação. </li></ol></li><li> Elimine o gel e recuperar membrana de nitrocelulose usando uma pinça de plástico na extremidade da membrana. </li></ol> 6. O bloqueio da membrana e Detecção <ol><li> Lavar membrana imediatamente com solução salina tamponada 1x com fosfato (PBS). Não deixe que a membrana secar. </li><li> Imergir a membrana em 50 mL de 3% de leite-PBS, tampão e o local de bloqueio numa rocker à temperatura ambiente durante 1 h. Após a incubação, descartar o tampão de bloqueio. </li><li> Mergulhe membrana na solução de anticorpo primário 1% de leite-PBS. Incubar a membrana na solução de anticorpo primário durante a noite num agitador rotativo a 4 ° C. Após a incubação, descartar a solução de anticorpo primário. Dilui-se os anticorpos primários para uma proporção de 1: 2000 de uma solução de anticorpo de tampão de bloqueio. Nota: Para as amostras provenientes de ratinhos, usamos UNC 222 de coelho anti-MUC5B e de cabra anti-Proteína Fluorescente Verde (GFP). Para amostras provenientes de seres humanos, usamos anticorpos primários H300 anti-MUC5B e 45M1 anti-MUC5AC. </li><li> membrana Lavar 3 vezes com PBS durante 10 min num agitador rotativo. </li><li> Imergir membrana em solução de anticorpo secundário 1% de leite-PBS. Dilui-se a anticorpos secundários para uma proporção de 1: 7500 de solução de anticorpo para tampão de bloqueamento e incubar à temperatura ambiente durante 1 hora num agitador rotativo (por exemplo, 700 anti-coelho e anti-cabra 680.). Proteger da luz através da incubação de uma co não transparententainer. Descartar a solução de anticorpo secundário após 1 h. </li><li> membrana Lavar 3 vezes com PBS durante 5 min. Enxágüe membrana com dH2O para remover o sal. </li><li> Leia a membrana de um sistema de fluorescência de infravermelhos de acordo com as instruções do fabricante. </li></ol>

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgG+in+cervicovaginal+mucus+traps+HSV+and+prevents+vaginal+herpes+infections.">IgG in cervicovaginal mucus traps HSV and prevents vaginal herpes infections.</a> Mucosal Immunol. 7 (5), 1036-1044 (2014).</li><li>Linden, S. K., Sutton, P., Karlsson, N. G., Korolik, V., McGuckin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins+in+the+mucosal+barrier+to+infection.">Mucins in the mucosal barrier to infection.</a> Mucosal Immunol. 1 (3), 183-197 (2008).</li><li>Thornton, D. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+analysis+of+mucin+core+proteins.">Mass spectrometric analysis of mucin core proteins.</a> Methods Mol Biol. 842, 67-79 (2012).</li><li>van der Post, S., Thomsson, K. A., Hansson, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+enzyme+approach+for+the+characterization+of+glycan+modifications+on+the+C-terminus+of+the+intestinal+MUC2mucin.">Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.</a> J Proteome Res. 13 (12), 6013-6023 (2014).</li><li>Thornton, D. J., Carlstedt, I., Sheehan, J. 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Evidence for a cysteine-rich sequence repeated within the molecule.</a> J Biol Chem. 272 (14), 9561-9566 (1997).</li><li>Sheehan, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+molecular+intermediates+in+the+assembly+pathway+of+the+MUC5AC+mucin.">Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin.</a> J Biol Chem. 279 (15), 15698-15705 (2004).</li><li>Ehre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressing+mouse+model+demonstrates+the+protective+role+of+Muc5ac+in+the+lungs.">Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs.</a> Proc Natl Acad Sci U S A. 109 (41), 16528-16533 (2012).</li><li>Livraghi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+and+lung+pathology+due+to+mucosal+surface+dehydration+in+{beta}-epithelial+Na++channel-overexpressing+mice:+role+of+TNF-{alpha}+and+IL-4R{alpha}+signaling,+influence+of+neonatal+development,+and+limited+efficacy+of+glucocorticoid+treatment.">Airway and lung pathology due to mucosal surface dehydration in {beta}-epithelial Na+ channel-overexpressing mice: role of TNF-{alpha} and IL-4R{alpha} signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment.</a> J Immunol. 182 (7), 4357-4367 (2009).</li><li>Martino, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ER+stress+transducer+IRE1beta+is+required+for+airway+epithelial+mucin+production.">The ER stress transducer IRE1beta is required for airway epithelial mucin production.</a> Mucosal Immunol. 6 (3), 639-654 (2013).</li><li>Nguyen, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+beta-blockers+attenuates+inflammation+and+mucin+content+in+a+murine+asthma+model.">Chronic exposure to beta-blockers attenuates inflammation and mucin content in a murine asthma model.</a> Am J Respir Cell Mol Biol. 38 (3), 256-262 (2008).</li><li>Papakonstantinou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPD+exacerbations+are+associated+with+pro-inflammatory+degradation+of+hyaluronic+acid.">COPD exacerbations are associated with pro-inflammatory degradation of hyaluronic acid.</a> Chest. , (2015).</li><li>Abdullah, L. H., Wolber, C., Kesimer, M., Sheehan, J. K., Davis, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+mucin+secretion+from+human+bronchial+epithelial+cell+primary+cultures.">Studying mucin secretion from human bronchial epithelial cell primary cultures.</a> Methods Mol Biol. 842, 259-277 (2012).</li></ol>

Os autores não têm qualquer conflito de divulgar.

O protocolo de mucina transferência de Western descrito neste vídeo combina técnicas convencionais utilizados em biologia molecular para separar e transferir grandes macromoléculas, tais como ADN, com técnicas normais para a detecção da proteína, isto é., Imunotransferência. A mesma técnica pode ser aplicada para estudar a biologia dos glicosaminoglicanos complexas, tais como a desagregação de alta peso molecular do ácido hialurónico 18. Embora esta técnica poderia ser utilizada numa ...

As mucinas são normalmente produzidos por superfícies mucosas que revestem as cavidades expostas ao ambiente exterior (por exemplo., Respiratório, digestivo, reprodutivo, extensões da superfície ocular), bem como os órgãos internos (por exemplo, pâncreas, vesícula biliar, glândulas mamárias). A presença destas glicoproteínas mantém a hidratação da superfície e forma uma barreira física contra os agentes patogénicos. Embora a produção de mucina é essencial para a saúde das mucosas, hiper-concentração de mucina e / ou as propriedades do muco aberrantes pode conduzir a obstrução do canal, a colonização bacteriana e inflamação crónica, o que pode causar danos irreversíveis. Uma cascata semelhante de eventos são observados em várias doenças, por exemplo., Fibrose cística 1, otite média crônica 2 e infecção cérvico-3. Portanto, é importante para entender o papel de mucinas na saúde e na doença e para estabelecer protocolos de rotina para a identificação de proteínas. A data, 19 genes mucinas foram identificados e codificam para cadeias polipeptídicas grandes que variam de 1.200 (por exemplo., MUC-1) a 22.000 (por exemplo, MUC16) aminoácidos. A família de genes de mucina pode ser dividido em dois subtipos: as mucinas associadas a membranas, envolvidas na sinalização celular e a superfície de blindagem, e as mucinas de formação de gel, responsáveis ​​pelas propriedades viscoelásticas dos geles de muco. mucinas associadas a membranas são principalmente monomérico e atribuem às superfícies das células através de um domínio que atravessa a membrana hidrofóbica. Em contraste, as mucinas de formação de gel possui vários domínios -como e rica em cisteína do factor de von Willebrand (vWF), que são essenciais para a formação de redes poliméricas dinâmicas. Grandes glicanos estão ligados a serina e treonina distribuídos por todo o apomucin. Estes oligossacáridos ligados em O densas pode contribuir-se para 80% do peso molecular 4. Intra- e inter-molecular de ligações dissulfureto que ligam os monómeros de mucina assegurar a integridade do gel de mucina network. Como resultado de glicosilação pesada e multimerização, mucinas encontram-se entre as moléculas maiores no mundo animal e não podem ser analisados ​​por electroforese em gel padrão através de gel de SDS-PAGE de poliacrilamida convencional e escadas de proteína padrão. Estes métodos / resolver proteínas separadas com pesos moleculares menores do que 250 kDa, enquanto os monómeros de mucina pode atingir até 2 MDa no caso de MUC16. No entanto, as escadas de proteína de alto peso molecular podem ser utilizados para estudar pequenas monómeros de mucina (isto é., A MUC1). Uma variedade de técnicas pode ser aplicado para estudar tamanho mucina, conformação e interacção. Tradicionalmente, a caracterização bioquímica das mucinas é realizado por mucina isolamento através de centrifugação isopícnica em gradiente de densidade em tampão de desnaturação, seguida de cromatografia de exclusão de tamanho e imunodetecção (por ex., Slot blotting) 5. e / ou dispersão de luz multi-ângulo dinâmico fornecer informações sobre o estado oligomérico de amostras ricas em mucina &lt;sup&gt; 1. Além disso, uma centrifugação zonal acoplado com imunodetecção e microscopia electrónica de transmissão são comumente usados ​​para determinar a conformação de macromoléculas de mucinas 6. A espectrometria de massa é também utilizado para quantificar as mucinas, detectar a clivagem proteolítica e analisar a composição 1,7,8 oligossacárido. Tais técnicas são dispendiosas, demoradas e frequentemente exigem grandes volumes e / ou altas concentrações de amostra. A metodologia aqui descrita, isto é., Mucina separação por electroforese, é reprodutível, de baixo custo e pode ser utilizado em estudos de alto rendimento para fornecer quantificação relativa e mucina investigar montagem polímero. No entanto, este ensaio necessita de alta afinidade, anticorpos de mucina de alta especificidade que pode não estar disponível para mucinas raras (ex., MUC19) ou certas espécies (por exemplo., Porco, furão). Western blotting de agarose é adequado para resolver uma grande variedade de amostras ricas em mucina com Concentrações que variam de 50 ug / ml (por exemplo., células de lavagens) a 5 mg / ml (por exemplo., expectoração). Este ensaio foi introduzida nos anos 1990 e só foi efectuado em alguns laboratórios especializados 9,10. Inicialmente, esta técnica permitiu identificar subpopulações de monómeros de mucina nas secreções respiratórias humanas 11,12 e confirmou o processo de oligomerização de células caliciformes, que consiste de formação de dímeros no retículo endoplasmático seguido por dímero de multimerização no aparelho de Golgi 13. Mais recentemente, a geração de anticorpos policlonais contra mucinas murino facilitou os estudos em modelos animais pequenos (por exemplo., Mucina modelos de ratos deficientes, βENaC, OVA-desafiado) e abriu um novo campo de pesquisa para estudos pré-clínicos compostos de teste farmacológico destinadas a remover o muco os pulmões 14-17. Como resultado de um crescente interesse em biologia mucina e a geração de novos, os anticorpos de mucina mais específicos, descrevemos herein a metodologia para separar mucinas por electroforese em gel de agarose, transferência para nitrocelulose e vácuo de duas cores detecção fluorescente no infravermelho.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>T6060-1kg</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma</td>
			<td>ARK2183-1L</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS-100g</td>
			<td>100g</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma</td>
			<td>114391-5g</td>
			<td>5g</td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>Sigma</td>
			<td>L6026-50g</td>
			<td>50g</td>
		</tr>
		<tr>
			<td>20X SSC (Sodium Saline Citrate) Buffer</td>
			<td>Promega</td>
			<td>V4261</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-1</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Trisodium Citrate (Na3C6H5O7)</td>
			<td>Sigma</td>
			<td>W302600-1kg</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>10 mM DTT (dithiothreitol)&#160;</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Milk Powder</td>
			<td>Saco</td>
			<td></td>
			<td>Instant non-fat dry milk</td>
		</tr>
		<tr>
			<td>Dulbecco Phosphate Buffered Saline (D-PBS)</td>
			<td>Gibco lifetechnologies</td>
			<td>14200-025</td>
			<td>500mL</td>
		</tr>
		<tr>
			<td>Anti-GFP Goat Primary Antibody (mouse samples)</td>
			<td>Rockland antibodies and assays</td>
			<td>600-101-215</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>UNC 222 Anti-Muc5b Rabbit Primary Antibody (mouse samples)</td>
			<td>UNC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H300 Anti-MUC5B Primary Antibodies (human samples)</td>
			<td>Santa Cruz</td>
			<td>sc-20119</td>
			<td>200ug</td>
		</tr>
		<tr>
			<td>45M1 Anti-MUC5AC Primary Antibodies (human samples)</td>
			<td>Abcam</td>
			<td>ab3649</td>
			<td>100ug</td>
		</tr>
		<tr>
			<td>Donkey Anti-rabbit 800CW IR Dye</td>
			<td>LI-COR Biosciences</td>
			<td>926-32213</td>
			<td>0.5mg</td>
		</tr>
		<tr>
			<td>Donkey Anti-mouse 680LT IR Dye</td>
			<td>LI-COR Biosciences</td>
			<td>926-68022</td>
			<td>0.5mg</td>
		</tr>
		<tr>
			<td>Electrophoresis Gel Box and Casting Tray</td>
			<td>Owl Seperation Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply Box</td>
			<td>Biorad</td>
			<td>Model 200/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Blotting Paper</td>
			<td>Amersham&#160;</td>
			<td>10600016&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Paper and Nitrocellulose membrane&#160;</td>
			<td>GE&#160;</td>
			<td>10 439 196</td>
			<td></td>
		</tr>
		<tr>
			<td>Boekel/Appligene Vacuum Blotter 230v</td>
			<td>Expotech USA</td>
			<td>230600-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Infrared Fluorescence System</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mucin agarose gel electrophoresis: western blotting for high-molecular-weight glycoproteins

Mucinas, as proteínas altamente glicosiladas que revestem as superfícies das mucosas, evoluíram como um componente chave da defesa inata, protegendo o epitélio contra patógenos invasores. O principal papel destas macromoléculas é facilitar aprisionamento de partículas e ao promover a lubrificação depuração da mucosa. Durante a síntese de proteínas, mucinas submetidos a intensa O-glicosilação e multimerização, o que aumenta drasticamente a massa e tamanho destas moléculas. Estas modificações pós-translacionais são essenciais para as propriedades viscoelásticas do muco. Como um resultado da natureza bioquímicas e biofísicas complexo destas moléculas, que trabalha com mucinas fornece muitos desafios que não podem ser superadas por meio de métodos convencionais de análise de proteína. Por exemplo, o seu elevado peso molecular evita a migração electroforética através de géis de poliacrilamida regulares e a sua natureza pegajosa promove a adesão a uma tubagem experimental. No entanto, a investigar o papel de mucinas na saúde(Por ex., Manter a integridade da mucosa) e doenças (por ex., Hiper-concentração, mucostasis, cancro) recentemente ganhou interesse e mucinas estão a ser investigados como um alvo terapêutico. Uma melhor compreensão da função e produção de mucina macromoléculas pode conduzir a novas abordagens farmacêuticas, por exemplo, inibidores da exocitose da mucina do grânulo e / ou agentes mucolíticos. Portanto, protocolos consistentes e confiáveis ​​para investigar a biologia mucina são fundamentais para o avanço científico. Aqui, descrevemos métodos convencionais para separar macromoléculas de mucina por electroforese usando um gel de agarose, a transferência de proteínas para uma membrana de nitrocelulose, e detectar o sinal com anticorpos específicos de mucina, bem como leitor de gel fluorescente no infravermelho. Estas técnicas estão amplamente aplicável para determinar a quantificação de mucina, a multimerização e para testar os efeitos dos compostos farmacológicos sobre mucinas.

Nós mostramos resultados representativos de expressão de mucina a seguir a electroforese em gel de agarose na LBA dos pulmões de ratinhos (Figura 1). Neste exemplo, utilizou-se o gel de agarose a mostrar sobre-regulação de produção de mucina na sequência de IL-13 de tratamento do modelo de ratinho Tg-MUC5AC. O Western blot mostra uma representação visual de expressão de mucina, o qual pode ser usado para a análise quantitativa de multímero ou intensidade de ...

Watch this Scientific Journal Video about Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins at JoVE.com

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucina Agarose Gel Eletroforese: Western Blotting para glicoproteínas alta peso molecular


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

mucin-agarose-gel-electrophoresis-western-blotting-for-high-molecular

Research

JoVE Journal

Biology

25.2K visualizações.  University of North Carolina at Chapel Hill. Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly. 

Vídeo: Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

Eletroforese em Gel de gradiente desnaturante (DGGE)

Biologia

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blotting Usando o Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Avaliar motilidade celular Neural Stem Usando um dispositivo Agarose Gel baseado microfluídicos

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Triagem para Agregação de amilóide por Semi-desnaturação Eletroforese Gel Detergente-Agarose

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Desnaturação Uréia Eletroforese em Gel de Poliacrilamida (PAGE Urea)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extração de DNA genômico de alto peso molecular de solos e sedimentos

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Eletroforese em Gel de Poliacrilamida azul Nativo (BN-PAGE) para Análise de Complexos Multiproteicos de Lisados ​​Celular

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Visualização dos Intermediários UV-induzida replicação em E. coli Usando bidimensional Análise Agarose-gel

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blotting: Preparação de amostras para detecção

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Extração de DNA de alto peso molecular de Esteiras Microbianas

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

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