Damos las gracias a Zehavit Frenkel y Tolchinsky Sandra de asistencia técnica. La investigación relacionada con este trabajo es apoyado por becas de la Fundación Ciencia Israel (1229-1207) y el alemán-israelí Proyecto de Cooperación (DIP-DFG).

El protocolo está destinada para el análisis de cadenas de azúcares de las glicoproteínas o total de una glicoproteína de interés específico. El procedimiento es básicamente el mismo en ambos casos con algunas modificaciones como se indica a través del protocolo. <ol><li> Para el etiquetado del metabolismo de N-glicanos ligados uno tiene que usar una cultura subconfluentes de células de mamífero cultivadas durante la noche en una placa de 90 mm para cada muestra (muestras pueden representar diferentes puntos de tiempo o de tratamientos). Este protocolo está optimizado para las células NIH 3T3. </li><li> Comer a las células de la glucosa por la incubación en 5 ml de recién preparada previamente calentada (37 ° C) de glucosa en un medio libre, se suministra con dializados 10% de FCS y 4 mM piruvato sódico durante 5-15 minutos. </li><li> Vuelva a colocar el medio del hambre con 1 ml de pre-calentado (37 ° C) de glucosa en un medio libre con 400 Ci de [2 - 3 H] marcada con manosa (menor concentración de 65 Ci / ml se utiliza para el etiquetado de las glicoproteínas total), e incubar las células en las condiciones de cultivo de tejido normal durante 1 hora. </li><li> Eliminar el medio de etiquetado de cada muestra, y con cuidado añadir 2 ml de PBS a 4 ° C para las muestras correspondientes a pulso y colocarlos en hielo (estas muestras se denominan pulsos), mientras que las muestras correspondientes a la persecución que ser enjuagados tres veces con 2 ml de pre-calentado (37 ° C) medio normal de cultivo completo, y luego se colocan en una incubadora de CO 2 a 37 ° C con 5 ml de pre-calentado medio habitual para los períodos de persecución deseado. </li><li> Enjuague las muestras de pulso (previamente colocado en hielo) 3 veces con 2 ml de helado de PBS. Las células se raspa el plato en 2 ml de PBS utilizando un rascador de células, y se coloca en un tubo Eppendorf de 2 ml. </li><li> Corto giro de las células a 16000xg (06.10 segundos), descartar el sobrenadante y congelar el sedimento celular a -80 ° C. </li><li> Siga los pasos 5 y 6 también para las muestras de persecución en el final del tiempo de persecución. </li><li> Eliminar las células del congelador y lisar ellos por la adición de 300 l de tampón A, vortex brevemente e incubar durante 20 min en hielo, o en el caso del análisis de las glicoproteínas total, por incubación de 20 minutos sobre el hielo con tampón B, seguido por ultrasonidos (4 tiempos, 10 segundos, con un máximo de amplitud), luego hervir las muestras durante 5 minutos. </li><li> Centrifugar los lisados ​​a 16000xg durante 20 min a 4 ° C. Transferir el sobrenadante a un nuevo tubo Eppendorf y desechar el sedimento. </li><li> Si el análisis de las glicoproteínas total, proceder a la filtración molecular y deglicosilación (paso 17). Para la inmunoprecipitación de la glicoproteína H2a demostrado aquí, agregar 20l/sample de (Repligen) la proteína A-agarosa 1:01 suspensión y 3 l / muestra de nuestro conejo policlonal de anticuerpos anti H2a para la glicoproteína deseada (en nuestro ejemplo anticuerpos anti-H2a ), el sobrenadante del paso 9. </li><li> Incubar las muestras de 04.16 horas a 4 ° C, mezclando constantemente por la rotación lenta. </li><li> De girar las bolas a 16000xg durante 30 segundos y retirar con cuidado el sobrenadante con vacío. </li><li> Aclarar las cuentas, mediante la adición de 500 l de amortiguación D y agitación. Giro como en el paso 12 y eliminar el sobrenadante. Repita el lavado 3 veces. </li><li> Añadir 10μl desnaturalización de amortiguación (suministrado con el NEB endo-H kit) a la bolita de cuentas y hervir las muestras durante 5 minutos (cuando las proteínas totales se analizan, el deglicosilación se lleva a cabo en el filtro Microcon, paso 17). </li><li> De girar las bolas (16000xg durante 30 segundos), y la transferencia del sobrenadante en un nuevo tubo Eppendorf. Luego 0.5μl de endo H enzima se añaden a cada muestra, junto con 10 l de tampón de reacción (también suministrado con el kit NEB endo H), y las muestras se incuban a 37 ° C durante 3 horas. </li><li> Para separar los glicanos liberados de las proteínas, la muestra se diluye 5 veces con agua destilada y se coloca en la parte superior de un filtro molecular (Microcon Ultracel YM30), con un 30kDa de corte, luego se centrifuga a 14000Xg durante 3 min. Aplicar 50μl de agua destilada y repetir la centrifugación de las muestras. Este lavado se repite 2 veces más, manteniendo el flujo continuo. </li><li> Si usted está analizando glicoproteínas total, aplicar el sobrenadante de los lisados ​​(desde el paso 9) para el filtro Microcon. Lavar el extracto con 100 l de buffer E, 3 veces por centrifugación repetida en 14000Xg durante 3 min. Añadir 3 l de tampón de reacción 10X endo H y 1,5 l de la enzima endo H al retenido Microcon, y se incuba durante 3 horas a 37 º C. Los glicanos liberados se eluyó con agua desionizada por tres centrifugaciones repetidas, como en el paso 16. </li><li> Si lo desea, retenidos en los pasos 16 y 17, con endo H resistente a las glicoproteínas son incubados con 200mU de F N-glucosidasa en 15 l de tampón C a 37 ° C durante 16 h. Elución con agua destilada se hace como en el paso 16. </li><li> Coloque el tubo que contiene la reacción endo H de flujo continuo (medidas 16 y 17) en un concentrador de SpeedVacY secar las muestras por completo (esto se puede hacer hasta 45 ° C para acelerar este proceso). </li><li> Para preparar las muestras para el HPLC, volver a suspender las bolitas secas en 12 l del solvente HPLC (acetonitrilo: agua, 60:40, 1% de ácido fosfórico). </li><li> Ajustar el dispositivo HPLC (conectado a la columna Spherisorb) a un flujo constante del disolvente (1 ml / min) y presión (un valor en particular entre 1000 y 2000 psi), y colocar un colector de fracciones para cambiar un tubo de cada 1 minuto. </li><li> Cargar las muestras en el dispositivo HPLC e iniciar el colector de fracciones de forma simultánea. </li><li> Recoger 48 fracciones de 1 ml de cada corrida de muestras y de una carrera de una mezcla de oligosacáridos estándar. </li><li> Transferencia de 500 l de cada fracción a un vial de centelleo, y mezclar el contenido con 3 ml de líquido de centelleo soluble en agua. </li><li> La carga de los viales y leer en un contador de centelleo. </li><li> La lectura de la CPM se representa gráficamente como una función del número de fracciones. </li><li> Los valores de cpm en un pico de representar a una especie específica de glicanos de la siguiente manera: las fracciones 18-21 corresponden a M5, M6 a las fracciones 22-26, 27-31 fracciones de M7, M8 a las fracciones 32-35, 36-39 fracciones de M9, fracciones 40-42 a G1M9 y las fracciones 43-45 a G2M9. </li><li> La suma de los valores de CPM para las fracciones dentro de cada pico refleja la cantidad absoluta de una especie de glicanos específicos. Con el fin de compensar el hecho de que las especies que contienen más residuos de manosa adquirir más la etiqueta, la &quot;cantidad relativa molar&quot; de cada especie glicano se calcula como sigue: la cantidad absoluta de cada especie glicano se divide por el número de residuos de manosa que contiene . Este valor se divide por la suma de las cantidades molares relativas de todas las especies de glicano obtenidos para obtener el &quot;por ciento del total&quot; para cada especie de glicanos específicos. </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> Lanzado con endo H (cpm) una </td><td> 90000 </td><td> 16800 </td></tr><tr><td> Lanzado con N-glucosidasa F (cpm) b </td><td> 20400 </td><td> 3900 </td></tr><tr><td> Glicoproteínas en retenido (cpm) c </td><td> 371700 </td><td> 127695 </td></tr><tr><td> Dolichol-oligosacáridos en retenido (cpm) d </td><td> 4350 </td><td> 540 </td></tr><tr><td> Dolichol-oligosacáridos en retenido (%) e </td><td> 1,4 ± 0,7 </td><td> 0,2 ± 0,2 </td></tr></tbody></table> Tabla 1. Análisis de la liberación de las cadenas de azúcar en la N-vinculada con deglycosidases y de la presencia de dolichol-oligosacáridos en las muestras de la glicoproteína. Se aplicó el procedimiento de pulso perseguir a un lisado de células NIH 3T3. Después de la filtración Microcon, denominado N-oligosacáridos fueron puestos en libertad, principalmente con endo H después del pulso de etiquetado (tipo manosa alta) y con un tratamiento adicional con F N-glucosidasa después de un período de persecución, que corresponden al tipo complejo (Golgi procesados ​​glicanos), endo H resistente oligosacáridos. Este análisis cuantitativo determinó que dolichol-oligosacáridos, representaron una fracción insignificante de la etiqueta en el producto retenido inicial. Notas: <ol class="lalpha"><li> Células NIH 3T3 fueron pulso marcado con [2 - 3 H] manosa y persiguió con medio completo. Después de la lisis celular y la filtración a través de Microcon YM30 de alta manosa N-oligosacáridos fueron puestos en libertad de retenidos por la incubación con endo H. </li><li> Endo H-resistente material retenidos Microcon de (a) se incubó con F N-glucosidasa y puesto en libertad oligosacáridos se mide en un contador de centelleo. </li><li> Retenidos Microcon obtenido como en (a) antes del tratamiento endo H, se extrajeron cuatro veces con cloroformo: metanol: agua 10:10:03 para eliminar glicolípidos precursor, y el material no extraído disuelve en NaOH 1N y se cuentan en un contador de centelleo. </li><li> Dolichol-oligosacáridos fueron purificados a partir de extractos de (c) como se describe en 1 y en un contador de centelleo. </li><li> Promedio de 3 por ciento de los experimentos de dolichol-oligosacáridos (d) del total de cpm en retenido (c + d). </li></ol> Resultados representante <img src="/files/ftp_upload/1899/1899fig1.jpg" alt="Figura 1" /> Figura 1. Pulso-caza análisis del total de las glicoproteínas NIH 3T3 en células no tratadas y la inhibición proteasomal Después de 1 hora de pulso con el etiquetado. [2 - 3 H] se obtuvo un perfil de espera con algunos glucosiladaprecursores restantes (Glc Man 9 GlcNAc 2 2 (G2M9) y G1M9), pero la mayor parte de la etiqueta presente en M9 y M8 especies, siendo este último el resultado de la poda de todos los niveles de glucosa y un residuo de manosa (Figura 1 A). No precursores libres pequeña manosa o de otro tipo se han detectado, lo que demuestra la minuciosidad de la purificación. Después de una larga persecución en vez 8h había más extensa de recorte para M7-6 y una pequeña cantidad de M5, pero las principales especies sigue siendo M9 y M8 (Figura 1 B). Si la persecución se hizo en presencia de un inhibidor de la proteasomal (30 M MG-132), sólo había una acumulación de menor importancia de estas mismas especies recortadas (Figura 1 C). <img src="/files/ftp_upload/1899/1899fig2.jpg" alt="Figura 2" /> Figura 2. El análisis cuantitativo de las cadenas de azúcar de un sustrato ERAD en comparación con las glicoproteínas total. (A) En un experimento similar a la de la Figura 1, en ​​relación cantidades molares de cada especie de oligosacáridos se calcularon con base en el contenido de manosa. Hemos convertido los valores de cpm obtenidos para cada especie de glicanos, el porcentaje de cada especie en relación con la suma total de las cantidades molares relativas de todas las especies presentes se representó posteriormente en función del tiempo de persecución por un promedio de dos experimentos. (B) similares (A), excepto que glicanos liberados de la ERAD sustrato ASGPR H2a fueron analizadas después de que el etiquetado de pulso y caza de hasta 4 horas, en la presencia o ausencia del inhibidor de la proteasomal MG-132. (C) Los valores de 4 horas de persecución en la presencia de MG-132 de (A) y (B) se comparan para ASGPR H2a y la piscina de la glicoproteína. (D) Esquema de la cadena de azúcar unidos a N procesos de corte en la sala de emergencias. Azúcar recortar los procesos que conducen a la M6-5 ligada a las proteínas (R) que se dirigen a ERAD en comparación con el M9-8 en los que salen al aparato de Golgi y más allá. Los resultados indican que el proceso de ERAD se asocia con la manosa corte de la N-glicanos para producir especies con 5-6 residuos de manosa 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>

El análisis del pulso-caza de los glicanos en las células vivas con una separación de HPLC proporciona un método para el estudio de la dinámica de los oligosacáridos alteraciones estructurales a lo largo de la vida de una glicoproteína. Hay una creciente evidencia de que tales alteraciones están involucrados en la producción de las señales para doblar ER, control de calidad y sistemas de tráfico 2-5. El método se puede aplicar no sólo para una glicoproteína de interés específico, sino también...

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		<th>Material Name</th>
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		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
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<td>Frac-100 fraction collector</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>18-1000-77</td>
<td>&nbsp;</td>
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<td>LS 6500 Liquid Scintillation Counting systems</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>510720</td>
<td>&nbsp;</td>
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<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>
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<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>
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<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>
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pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

La unión de la Glc<sub&gt; 3</sub&gt; Hombre<sub&gt; 9</sub&gt; GlcNAc<sub&gt; 2</subPrecursor&gt; oligosacáridos a polipéptidos nacientes en la sala de emergencia es una modificación común para proteínas secretoras. A pesar de esta modificación fue implicado en numerosos procesos biológicos, otros aspectos de su función están surgiendo, con la evidencia reciente de su papel en la producción de señales para el control de la calidad de la glicoproteína y la trata. Por lo tanto, fenómenos relacionados con la N-glicanos ligados y su tratamiento están siendo intensamente investigados. Los métodos que se han desarrollado recientemente para el análisis de proteómica han mejorado mucho la caracterización de la glicoproteína N-glicanos ligados. Sin embargo, no proporcionar información sobre la dinámica de la cadena de elaboración de azúcar en cuestión. Para ello, el etiquetado y el pulso-caza protocolos de análisis que se utilizan suelen ser complejos y dan rendimientos muy bajos. Se describe aquí un método sencillo para el aislamiento y el análisis de metabólicamente etiquetados N-oligosacáridos. El protocolo se basa en el etiquetado de las células con [2 -<sup&gt; 3</sup&gt; H] manosa lisis, la desnaturalización y la liberación enzimática de los oligosacáridos de cualquiera de una proteína específica immunoprecipitated de interés o de la piscina glicoproteína en general por los tratamientos secuenciales con endo H y N-glucosidasa F, seguido por filtración molecular (Amicon). En este método, los oligosacáridos aislados servir como insumo para el análisis por HPLC, que permite la discriminación entre las diversas estructuras de glicanos en función del número de unidades de monosacáridos que los componen, con una resolución de un monosacárido individual. El uso de este método hemos sido capaces de estudio de alta manosa unidos a N perfiles de oligosacáridos de glicoproteínas total de células después del pulso de la Caza en condiciones normales y en la inhibición del proteosoma. Estos perfiles fueron comparados con los obtenidos de una ER-immunoprecipitated asociados degradación (ERAD) sustrato. Nuestros resultados sugieren que la mayoría de los celulares NIH 3T3 glicoproteínas son relativamente estables y que la mayoría de los oligosacáridos se recortan para el hombre<sub&gt; 9.8</sub&gt; GlcNAc<sub&gt; 2</sub&gt;. En contraste, los sustratos inestables ERAD se recortan para el hombre<sub&gt; 5.6</sub&gt; GlcNAc<sub&gt; 2</sub&gt; Y glicoproteínas teniendo estas especies se acumulan en la inhibición de la degradación proteasomal.

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

Se describe un método para el análisis de la alteración de la N-glicanos ligados a través de los primeros años de vida de las glicoproteínas después de su biosíntesis en células de mamíferos. Esto se logra mediante pulso-caza análisis de glicanos metabólicamente etiquetados, liberación enzimática de las glicoproteínas y análisis por HPLC.

Pulso-caza de análisis de cadenas de azúcares unidos a N de glicoproteínas en células de mamífero

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 vistas.  Tel Aviv University. Se describe un método para el análisis de la alteración de la N-glicanos ligados a través de los primeros años de vida de las glicoproteínas después de su biosíntesis en células de mamíferos. Esto se logra mediante pulso-caza análisis de glicanos metabólicamente etiquetados, liberación enzimática de las glicoproteínas y análisis por HPLC.

Video: 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.

En Fraccionamiento subcelular situ de células mamíferas adherentes y no adherentes-

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

Investigación

Biología

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.

El análisis de ADN de doble cadena Break (OSD) de reparación en células de mamífero

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.

MISIÓN esiRNA sobre el cribado del ARNi en células de mamífero

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.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

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.

Análisis de la cinética de exportación nuclear de ARNm en células de mamífero por microinyección

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.

Análisis de la posición del ciclo celular en células de mamífero

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.

Análisis de Expresión de mamíferos enlazador histonas 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.

La visualización de los ARNm retículo endoplásmico localizados en 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.

Inducción y Análisis de epitelial a mesenquimal de transición

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.

Medición de niveles hemo síntesis en células de mamífero

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 ...