Agradecemos a Zehavit Frenkel e Sandra Tolchinsky para assistência técnica. Pesquisas relacionadas a este trabalho é suportado por concessões do Ciência Israel Foundation (1229-1207) eo alemão-israelense Projeto de Cooperação (DIP DFG).

O seguinte protocolo destina-se a análise de cadeias de açúcar de glicoproteínas total ou de uma glicoproteína específica de interesse. O procedimento é basicamente o mesmo para ambos os casos, com algumas alterações como indicado ao longo do protocolo. <ol><li> Para a rotulagem metabólica de N-glicanos ligados a pessoa precisa usar uma cultura confluentes de células de mamíferos cultivadas durante a noite em um prato de 90 mm para cada amostra (amostras podem representar diferentes momentos ou tratamentos). Este protocolo é otimizado para as células NIH 3T3. </li><li> Morrer de fome as células para a glicose através da incubação em 5 ml de recém-preparados médio pré-aquecido (37 ° C), glicose-free, fornecido com 10% FCS dialisadas e 4 mM piruvato de sódio por 5-15 minutos. </li><li> Substituir o meio de fome com 1 ml da pré-aquecido (37 ° C) sem glicose meio contendo 400 μCi de [2-3 H] marcado manose (menor concentração de 65 μCi / ml é utilizado para a rotulagem de glicoproteínas total), e incubar as células do tecido normal condições de cultura para uma hora. </li><li> Remova a mídia de rotulagem de cada amostra e, cuidadosamente, adicionar 2 ml de PBS a 4 ° C para as amostras correspondentes a pulso e coloque-os sobre gelo (estas amostras são referidos como pulsos), enquanto as amostras correspondentes ao assunto precisa ser lavado 3 vezes com 2 ml de pré-aquecido (37 ° C) meio de cultura normal, completa, e depois colocados em uma incubadora de CO 2 a 37 ° C com 5 ml de pré-aquecido médio regular para o período desejado perseguição. </li><li> Lave as amostras de pulso (previamente colocado no gelo) 3 vezes com 2 ml de PBS gelado. As células são então raspado o prato em 2 ml de PBS com uma espátula de células, e colocado em um tubo Eppendorf 2 ml. </li><li> Curto girar a células em 16000Xg (6-10 seg), desprezar o sobrenadante e congelar o pellet celular a -80 ° C. </li><li> Execute os passos 5 e 6 também para as amostras de perseguição no final do tempo de perseguição. </li><li> Remove as células do congelador e lisar-los através da adição de 300 l de tampão A, brevemente vórtex e incubar por 20 min no gelo, ou no caso de análise de glicoproteínas total, por 20 min de incubação no gelo com tampão B, seguidos por sonicação (4 vezes, 10 segundos, máxima amplitude), em seguida, ferver as amostras para 5min. </li><li> Centrifugar a lisados ​​em 16000Xg por 20 min a 4 ° C. Transfira o sobrenadante para um tubo Eppendorf novo e descartar o pellet. </li><li> Se a análise de glicoproteínas total, proceder à filtração molecular e deglycosylation (passo 17). Para imunoprecipitação da glicoproteína H2a demonstrado aqui, adicione 20l/sample de (Repligen) proteína A-agarose contas 01:01 de suspensão e 3 mL / amostra do nosso coelho anticorpo policlonal anti H2a para a glicoproteína desejado (no nosso exemplo de anticorpos anti-H2a ), para o sobrenadante do passo 9. </li><li> Incubar as amostras para 4-16 horas a 4 ° C, em constante mistura por rotação lenta. </li><li> Spin para baixo as contas em 16000Xg por 30 segundos, e remova cuidadosamente o vácuo sobrenadante usando. </li><li> Lave as contas, por adição de 500 mL de buffer D e vórtice. Rotação como no passo 12 e remover o sobrenadante. Repetir a lavagem três vezes. </li><li> Adicionar 10μl tampão de desnaturação (fornecido com o endo-H NEB kit) para o pellet talão e ferva as amostras por 5 min (quando as proteínas totais são analisados, a deglycosylation é realizado no filtro microcontrolador, passo 17). </li><li> Girar as esferas (16000Xg por 30 segundos), e transferir o sobrenadante para um tubo Eppendorf novo. Então 0.5μl da enzima endo H são adicionados a cada amostra, juntamente com 10 ml do tampão de reação (também fornecido com o kit H endo NEB) e as amostras são incubadas a 37 ° C por 3 horas. </li><li> Para separar os glicanos liberada a partir das proteínas, a amostra é diluída 5 vezes com água deionizada e colocado em cima de um filtro molecular (microcontrolador Ultracel YM30), com um corte de 30kDa, em seguida, centrifugado a 14000Xg por 3 min. Aplicar 50μl de água deionizada e repetir a centrifugação das amostras. Esta lavagem é repetida vezes mais 2, mantendo o escoamento. </li><li> Se você está analisando glicoproteínas total, aplicar o sobrenadante do lisados ​​(a partir do passo 9) para o filtro microcontrolador. Lavar o extrato com 100 l de tampão E, 3 vezes por centrifugação repetida em 14000Xg por 3 min. Adicionar 3 mL de tampão de reação 10X endo H e 1,5 mL da enzima endo H ao retentado microcontrolador, e incubar durante 3 horas a 37 ° C. Os glicanos liberados são eluídos com água deionizada por 3 centrifugações repetidas, como no passo 16. </li><li> Se desejar, retentados das etapas 16 e 17, contendo endo H-resistentes glicoproteínas são então incubadas com 200mU de N-glicosidase F em 15 mL de tampão C a 37 ° C por 16 h. Eluição com água deionizada é feito como na etapa 16. </li><li> Coloque o tubo que contém o endo H reação de fluxo (etapas 16 e 17) em um concentrador SpeedVac, E secar as amostras completamente (este pode ser feito até 45 ° C para acelerar esse processo). </li><li> Para preparar as amostras para a HPLC, ressuspender o pellets secos em 12 mL do solvente HPLC (acetonitrila: água, 60:40, ácido fosfórico 1%). </li><li> Ajustar o dispositivo de HPLC (conectado à coluna Spherisorb) para fluxo constante de solvente (1 ml / min) e pressão (um valor em particular entre 1000 e 2000 psi), e colocar um coletor de frações para mudar um tubo a cada 1 minuto. </li><li> Carregar as amostras no dispositivo HPLC e iniciar o coletor de fração ao mesmo tempo. </li><li> Coletar 48 frações de 1 ml de cada amostra e executar de uma corrida de uma mistura padrão de oligossacarídeos. </li><li> Transferência de 500 mL de cada fração para um frasco de cintilação, e misturar o conteúdo com 3 ml de água-miscível líquido de cintilação. </li><li> Carregar os frascos e lido em um contador de cintilação. </li><li> A leitura cpm é plotado como uma função do número fracionário. </li><li> Os valores cpm dentro de um pico de representar uma espécie específica glicano como segue: frações 18-21 correspondem a M5, M6 para frações 22-26, 27-31 frações de M7, frações 32-35 a M8, frações 36-39 a M9, frações 40-42 para 43-45 G1M9 e frações de G2M9. </li><li> A soma dos valores cpm para as frações dentro de cada pico reflete a quantidade absoluta de uma espécie glicano específico. , A fim de compensar o fato de que as espécies que contenham mais resíduos de manose adquirir mais etiqueta, a &quot;quantidade relativa molar&quot; de cada espécie glicano é calculado da seguinte forma: a quantidade absoluta de cada espécie glicano é dividido pelo número de resíduos de manose que contém . Este valor é então dividido pela soma dos montantes relativos molar de todas as espécies glicano obtidos para obter o &quot;por cento do total&quot; para cada espécie glicano específico. </li></ol><table border="1"><tbody><tr><td style="text-align:center;"> Chase (h) </td><td style="text-align:center;"> 0 </td><td style="text-align:center;"> 4 </td></tr><tr><td> Lançado com endo H (cpm) a </td><td> 90000 </td><td> 16800 </td></tr><tr><td> Lançado com N-glicosidase F (cpm) b </td><td> 20400 </td><td> 3900 </td></tr><tr><td> Glicoproteínas no retentado (cpm) c </td><td> 371700 </td><td> 127695 </td></tr><tr><td> Dolicol-oligossacarídeos no retentado (cpm) d </td><td> 4350 </td><td> 540 </td></tr><tr><td> Dolicol-oligossacarídeos no retentado (%) e </td><td> 1,4 ± 0,7 </td><td> 0,2 ± 0,2 </td></tr></tbody></table> Tabela 1. Análise da liberação de cadeias N-linked açúcar com deglycosidases e da presença de Dolicol-oligossacarídeos em amostras de glicoproteína. Foi aplicado o procedimento de pulso-caça a um lisado de células NIH 3T3. Após filtração microcontrolador, rotulado N-linked oligossacarídeos foram liberados principalmente com endo H após a rotulagem de pulso (tipo manose alta) e com tratamento adicional com N-glicosidase F após um período de perseguição, o que corresponderia a tipo complexo (Golgi-processados ​​glicanos), oligossacarídeos endo H resistente. Esta análise quantitativa determinou que Dolicol-oligossacarídeos, foi responsável por uma fração insignificante do rótulo no retentado inicial. Notas: <ol class="lalpha"><li> Células NIH 3T3 foram pulso marcado com [2-3 H] manose e perseguido com meio completo. Após a lise celular e filtração através de microcontrolador YM30, alta manose N-linked oligossacarídeos foram liberados de retentados de incubação com Endo H. </li><li> Endo material de H-resistentes em retentados microcontrolador a partir de (a) foi então incubada com N-glicosidase F e liberado oligossacarídeos medida em um contador de cintilação. </li><li> Retentados microcontrolador obtida como em (a) antes do tratamento endo H, foram extraídos quatro vezes com clorofórmio: metanol: água para remover 10:10:03 glycolipids precursor, eo material não extraído solubilizado em NaOH 1N e contados em um contador de cintilação. </li><li> Dolicol-oligossacarídeos foram purificados de extratos a partir de (c) como descrito em 1 e contados em um contador de cintilação. </li><li> Média de 3 experimentos para por cento do Dolicol-oligossacarídeos (d) de cpm total de retentado (c + d). </li></ol> Resultados representante <img src="/files/ftp_upload/1899/1899fig1.jpg" alt="Figura 1" /> Figura 1. Pulso-chase análise de glicoproteínas total de NIH 3T3 em células não tratadas e sobre a inibição proteosomal Após 1h de pulso com rotulagem. [2-3 H] obtivemos um perfil esperado com alguma glucosylatedprecursores restante (Glc Man 2 9 GlcNAc 2 (G2M9) e G1M9), mas a maioria do rótulo presente em M9 e M8 espécies, sendo este último o resultado do corte de toda a glicose e um resíduo de manose (Figura 1 A). No free manose ou outros pequenos precursores foram detectados, atestando a meticulosidade da purificação. Na sequência de um pouco longo 8h perseguição havia mais extenso corte para M7-6 e uma pequena quantidade de M5, mas as principais espécies continuaram a ser M9 e M8 (Figura 1 B). Se a perseguição foi feito na presença de um inibidor proteosomal (30 mM MG-132), havia apenas um acúmulo menor dessas espécies mesmo aparadas (Figura 1 C). <img src="/files/ftp_upload/1899/1899fig2.jpg" alt="Figura 2" /> Figura 2. Análise quantitativa de cadeias de açúcar de um substrato ERAD comparação com glicoproteínas total. (A) Em um experimento semelhante ao da Figura 1, relativa quantidade molar de cada espécie de oligossacarídeos foram calculados com base no conteúdo de manose. Convertemos os valores obtidos cpm para cada espécie glicano, a porcentagem de cada espécie em relação à soma total dos valores relativos molar de todos os presentes espécies foi então plotado como uma função do tempo de perseguição por uma média de dois experimentos. (B) Similar para (A), exceto que glicanos liberado do substrato ERAD ASGPR H2a foram analisados ​​após a rotulagem de pulso e de perseguição por até 4h, na presença ou ausência do inibidor proteosomal MG-132. (C) Os valores para 4h de perseguição na presença da MG-132 a partir de (A) e (B) são comparados para ASGPR H2a ea piscina glicoproteína. (D) Regime de N-linked cadeia de açúcar corte processos na ER. Processos de corte de açúcar que levam à M6-5 ligado às proteínas (R) que são direcionados para ERAD comparação com M9-8 sobre aqueles que sair para o Golgi e além. Os resultados indicam que o processo ERAD é associado com a manose corte de N-glicanos para produzir espécies com 5-6 resíduos de manose restantes.

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

A análise do pulso-chase dos glicanos em células vivas, com separação por HPLC fornece um método para estudar a dinâmica do oligossacarídeo alterações estruturais ao longo da vida de uma glicoproteína. Há evidências crescentes de que tais alterações estão envolvidos na produção dos sinais de ER dobrar, controle de qualidade e 2-5 sistemas tráfico. O método pode ser aplicado não só para uma glicoproteína específica de interesse, mas também para analisar a dinâmica da estrutura de glico...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>600E Multisolvent delivery System controller</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT062710</td>
<td>&nbsp;</td>
<tr>
<td>Acetonitrile LiChrosolv (gradient grade for liquid chromatography)</td>
<td>&nbsp;</td>
<td>Merck Bioscience</td>
<td>1.0003</td>
<td>&nbsp;</td>
<tr>
<td>Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC503024 or 4208</td>
<td>&nbsp;</td>
<tr>
<td>Concentrator 5301, incl. 48 x 1.5 / 2.0 ml fixed-angle rotor</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5301 000.016</td>
<td>&nbsp;</td>
<tr>
<td>Dialyzed Foetal Calf Serum</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-011-1A</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modi&#xFB01;ed Eagle's Medium</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen Cell Culture</td>
<td>41965039</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modi&#xFB01;ed Eagle's Medium - glucose free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D5030</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>EDTA disodium salt (Titriplex Iii)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>1084180250</td>
<td>&nbsp;</td>
<tr>
<td>Endo Hf Kit (1,000,000 units/ml)</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>p0703S</td>
<td>&nbsp;</td>
<tr>
<td>Foetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-001-1A</td>
<td>&nbsp;</td>
<tr>
<td>Frac-100 fraction collector</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>18-1000-77</td>
<td>&nbsp;</td>
<tr>
<td>LS 6500 Liquid Scintillation Counting systems</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>510720</td>
<td>&nbsp;</td>
<tr>
<td>Mannose, D-[2-3H(N)]- (Specific Activity: 15-30Ci/mmol)</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>NET570A</td>
<td>&nbsp;</td>
<tr>
<td>N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>474790</td>
<td>&nbsp;</td>
<tr>
<td>N-glycosidase F (1000 units/ml)</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11365177</td>
<td>&nbsp;</td>
<tr>
<td>N-octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>O3757</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 cells</td>
<td>&nbsp;</td>
<td>American Type Culture Collection (ATCC)</td>
<td>CRL-1658</td>
<td>&nbsp;</td>
<tr>
<td>Opti-Flour</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>6013199</td>
<td>&nbsp;</td>
<tr>
<td>Phosphoric acid solution ( 49-51%, for HPLC)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>79607</td>
<td>&nbsp;</td>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2714</td>
<td>&nbsp;</td>
<tr>
<td>Protein A-Sepharose beads</td>
<td>&nbsp;</td>
<td>Repligen</td>
<td>IPA300</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit polyclonal anti-H2 carboxy-terminal 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>30970</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Bio-RAD</td>
<td>161-0301</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>342483</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution (100mM)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Spherisorb NH2 Column, 5 &mu;m, 4.6 x 250 mm</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>PSS831115</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>BDH</td>
<td>306324N</td>
<td>&nbsp;</td>
<tr>
<td>Vibra-Cell ultrasonic processors VCX 750</td>
<td>&nbsp;</td>
<td>Sonics and Materials, Inc.</td>
<td>690-003</td>
<td>&nbsp;</td>
<tr>
<td>Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 stably expressing H2a 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer D: 0.5% Triton X-100, 0.25% w/v sodium deoxycholate, 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M3148</td>
<td>&nbsp;</td>		</tbody>
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pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Fixação do Glc<sub&gt; 3</sub&gt; Homem<sub&gt; 9</sub&gt; GlcNAc<sub&gt; 2</sub&gt; Precursor oligossacarídeo de polipeptídeos nascentes na ER é uma modificação comum para proteínas secretoras. Embora esta modificação foi implicado em vários processos biológicos, os aspectos adicionais de sua função estão surgindo, com a evidência recente de seu papel na produção de sinais para o controle de qualidade e glicoproteína tráfico. Assim, os fenômenos relacionados com a N-glicanos ligados e seu processamento estão sendo intensamente investigadas. Métodos que têm sido recentemente desenvolvidos para a análise proteômica têm melhorado bastante a caracterização da glicoproteína N-glicanos ligados. No entanto, eles não fornecem insights sobre a dinâmica da cadeia de processamento de açúcar envolvidos. Para isso, rotulagem e protocolos de pulso-chase análise são usados ​​que normalmente são complexos e dão rendimentos muito baixos. Descrevemos aqui um método simples para o isolamento e análise de metabolicamente marcados N-linked oligossacarídeos. O protocolo baseia-se na marcação das células com [2 -<sup&gt; 3</sup&gt; H] lise, desnaturação manose e liberação enzimática do oligossacarídeos a partir de qualquer uma proteína específica immunoprecipitated de interesse ou da piscina glicoproteína geral por meio de tratamentos seqüenciais com endo H e N-glicosidase F, seguido de filtração molecular (Amicon). Neste método, os oligossacarídeos isolados servir como insumo para HPLC análise, que permite a discriminação entre as várias estruturas glicano acordo com o número de unidades de monossacarídeo composto por eles, com uma resolução de um monossacarídeo único. Usando este método conseguimos estudar alta manose N-linked perfis de oligossacarídeos de glicoproteínas total de células após o pulso-chase em condições normais e sob inibição do proteassoma. Esses perfis foram comparados com aqueles obtidos a partir de uma ER associada à degradação do substrato immunoprecipitated (ERAD). Nossos resultados sugerem que a maioria dos NIH 3T3 glicoproteínas celulares são relativamente estáveis ​​e que a maioria de seus oligossacarídeos são aparados para Homem<sub&gt; 08/09</sub&gt; GlcNAc<sub&gt; 2</sub&gt;. Em contraste, substratos ERAD instáveis ​​são aparados para Homem<sub&gt; 05/06</sub&gt; GlcNAc<sub&gt; 2</sub&gt; E glicoproteínas tendo estas espécies se acumulam sobre a inibição da degradação proteosomal.

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Nós descrevemos um método para análise da alteração da N-glicanos ligados através do início da vida de glicoproteínas após a sua biossíntese em células de mamíferos. Isto é conseguido através de pulso-chase análise de glicanos metabolicamente marcados, a liberação enzimática das glicoproteínas e análise por HPLC.

Pulso-chase Análise de Cadeias N-linked Açúcar de Glicoproteínas em células de mamíferos

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

pulse-chase-analysis-n-linked-sugar-chains-from-glycoproteins

Research

JoVE Journal

Biology

15.1K visualizações.  Tel Aviv University. Nós descrevemos um método para análise da alteração da N-glicanos ligados através do início da vida de glicoproteínas após a sua biossíntese em células de mamíferos. Isto é conseguido através de pulso-chase análise de glicanos metabolicamente marcados, a liberação enzimática das glicoproteínas e análise por HPLC.

Vídeo: Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Fracionamento em Subcelulares situ de aderentes e não aderentes células de mamíferos

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Biologia

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Análise de DNA dupla fita-Break Repair (DSB) em células de mamíferos

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

EsiRNA MISSÃO para a seleção RNAi em células de mamíferos

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Análise de Cinética mRNA Nuclear Exportação em células de mamíferos por microinjeção

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análise da posição do ciclo celular em células de mamíferos

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Análise de Expressão de mamíferos Linker-histona Subtipos

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualização de mRNAs retículo endoplasmático localizados em células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medição dos Níveis de heme em células de mamíferos Síntese

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...