<html> 1. Déglycosylation enzymatique <ol><li> Utiliser des tubes de PCR afin de minimiser la perte d&#39;eau due à l&#39;évaporation. Repérer un ensemble de tubes 1 à 7. </li><li> Décongelez le tampon 10X G7, le tampon 10X glycoprotéine dénaturation, et les 10% NP-40 et tapotez doucement les tubes pour mélanger le contenu. Conserver à température ambiante. </li><li> Placer les flacons contenant des enzymes sur la glace. Essayez de minimiser le dégel / gel cycles. </li><li> Dissoudre le contenu du flacon hCGβ (150 ug) dans 600 pi de dH 2 O et de garder sur la glace. </li><li> Préparer 1 ml de tampon 1X G7 en diluant le stock 10X en DH 2 O. </li><li> Diluer 0,5 pl de la PNGase F dans 25 pi de tampon 1X G7 et de garder sur la glace. </li><li> Préparer le mélange exoglycosidase (mix EG) en combinant 2 pl chacune des α-N-acétylgalactosaminidase, α1-2 Fucosidase, β1-3 galactosidase, et α1-3, 6 galactosidase. </li><li> Mettre en place des tubes de PCR comme indiqué: </li></ol>ak »&gt; <table border="1"><tbody><tr><td> Tube / échantillon # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ (0.25mg/ml) </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> Glycoprotéine 10X tampon dénaturant </td><td> 1 microlitre </td><td> 1 microlitre </td><td> 1 microlitre </td><td> 1 microlitre </td><td> 1 microlitre </td><td> 1 microlitre </td><td> 1 microlitre </td></tr><tr><td> dH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li> Boucher les tubes, mélanger délicatement et placer dans lethermocycleur, fermer le couvercle et dénaturer les protéines par incubation de 10 minutes à 94 ° C, suivie d&#39;un 4 ° C détiennent (en utilisant des tubes PCR dans un thermocycleur empêche grandement l&#39;évaporation dans les échantillons de petit volume). </li><li> Retirer les tubes du thermocycleur et centrifugeuse pour éliminer toute condensation visible. </li><li> Ajouter les réactifs suivants comme indiqué: </li></ol><table border="1"><tbody><tr><td> Exemple # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10% de NP-40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> Tampon 10X G7 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5 &amp; mu; L </td><td> 2.5μl </td></tr><tr><td> PNGase F 01h50 dil. </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td></tr><tr><td> Mélanger déglycosylation </td><td> - </td><td> - </td><td> 2μl </td><td> 2μl </td><td> - </td><td> 2μl </td><td> 2μl </td></tr><tr><td> Mélangez EG </td><td> - </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td></tr><tr><td> dH 2 O </td><td> 10 ul </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> Vol total de la réaction. </td><td> 25 L μ </td><td> 25 L μ </td><td> 25 L μ </td><td> 25 L μ &lt;/ Td&gt; <td> 25 L μ </td><td> 25 L μ </td><td> 25 L μ </td></tr></tbody></table><ol start="12"><li> Fermer les tubes PCR utilisant des amorces de nouvelles (jeter celles qui sont utilisées car elles ne s&#39;adaptent pas correctement après un cycle d&#39;incubation). </li><li> Mélanger les tubes en tapotant délicatement 4 fois, puis tourner le contenu vers le bas. </li><li> Placer les tubes dans le thermocycleur et incuber à 37 ° C pendant 4 heures puis laisser refroidir les échantillons à 4 ° C. </li></ol> 2. SDS-PAGE des échantillons déglycosylé <ol><li> Préparer 130 ul de tampon frais 3X réduction de la charge de SDS en ajoutant 4 pi de TNT 1.25M. </li><li> Ajouter 12,5 ul de la mémoire tampon 3X préparés de chargement réduisant SDS à chaque échantillon. </li><li> Fermer les tubes avec des bouchons de nouvelles et tapotez doucement les tubes pour mélanger. </li><li> Incuber les tubes dans un thermocycleur à 94 ° C pendant 5 minutes puis refroidir à 4 ° C. </li><li> Charge 30 ul de chaque échantillon et 10 pi de la protéine de marqueur sur un 10-20% Tris-Glycine gel. Enregistrer le reste de l&#39;échantillon pour la partie 3.1. Charge 10 pi de marqueur protéique ColorPlus précolorés. </li><li> Electrophorèse gel à 130 volts à température ambiante jusqu&#39;à ce que le front se trouve près du bas du gel. </li><li> Lorsque le gel a fini, enlever le gel de la fonte et le placer dans une petite boîte en plastique avec assez de Coomassie bleuissement pour couvrir le gel. </li><li> Stain le gel pendant 1 heure sous agitation douce. </li><li> Lavez les trois moments de gel pendant 30 minutes dans 50 ml de solution Décolorer. </li><li> Enregistrez les images en utilisant un transilluminateur lumière blanche ou un scanner. Sinon, le gel peut être séché entre des feuilles de cellophane dans un cadre. </li></ol> 3. Pro-Q Emerald 300 pour la détection des protéines glycosylées de gels SDS-PAGE <ol><li> En parallèle avec le gel de 2,1), charge le reste des échantillons sur un 10-20% Tris-Glycine gel. Charge 10 &amp; mu; L de protéines marqueurs ColorPlus précolorés. </li><li> Alors que le gel est en marche dissoudre le réactif Pro-Q émeraude avec du DMF et de préparer le correctif stock, Laver et oxydantes des solutions pour l&#39;Emeraude de Pro-Q 300 tache suivant le manuel du produit fournie avec le kit. </li><li> Lorsque l&#39;électrophorèse est terminée, enlever le gel de fonte et le placer dans une boîte en plastique. </li><li> Fixer le gel en ajoutant 100 ml de la solution Fix et c&#39;est une nuit à température ambiante sous agitation douce. </li><li> Laver le gel avec 100 ml de la solution de lavage de 10 à 20 minutes à température ambiante sous agitation Gentile. Répéter le lavage avec la solution de lavage frais. </li><li> Oxyder les hydrates de carbone en incubant le gel avec une agitation douce pendant 30 minutes dans 25 ml de solution oxydante. </li><li> Laver le gel comme décrit dans l&#39;étape 3.5. </li><li> Alors que le gel est de préparer le lavage douce Pro-Q Emerald 300 tache en ajoutant 500 pl de la solution Pro-Q Emeraude réactif dissous 300 à l&#39;étape 3.2 à 25 ml decoloration tampon fourni dans le kit. </li><li> Stain le gel en ajoutant 25 ml de la tache préparé à l&#39;étape 3.8 et l&#39;incubation dans l&#39;obscurité sous agitation douce pendant 90 à 120 minutes. </li><li> Répétez les deux étapes de lavage décrite à l&#39;étape 3.5. </li><li> Enregistrer les images avec un transilluminateur d&#39;UV à 300nm. Utilisez le marqueur de 80 kDa, qui est étiqueté avec Pro-Q vert émeraude, le chevauchement de l&#39;image à une image UV lumière blanche du gel montrant l&#39;échelle précolorés. </li><li> Comparez les images du gel Coomassie teinté à l&#39;étape 2.10 avec le gel d&#39;Emeraude Pro-Q teinté de l&#39;étape 3.11. </li></ol> 4. Les résultats représentatifs Les changements dans la migration des protéines enzymatiques après déglycosylation sont présentés dans la figure 2. Comparer l&#39;échantillon de contrôle (panneau A, ligne 1) avec le traitement PNGase F (élimination des N-glycanes, piste 2), et Mix déglycosylation (PNGase F; ainsi endo-et exoglycosidases pour enlever O-glycanes, piste 3). Aucune autre réductionla taille est observée après digestion par des glycosidases supplémentaires (piste 4). Outre un changement de masse, les bandes deviennent plus nettes que les glycanes sont supprimés. Une bande fonctionnant sous le marqueur de 17 kDa (astérisque) représente l&#39;parfaitement déglycosylé hCGβ polypeptide (MW: 16 kDa). D&#39;autres bandes pourraient découler de déglycosylation incomplètes ou multiples à partir des protéines non identifiés présents dans l&#39;échantillon hCGβ (voir ligne 1). Lanes 5 à 7 (contrôles) montrent les bandes correspondant aux glycosidases. La coloration par le vert émeraude glycoprotéine est montré dans le panneau B. Ce réactif s&#39;oxyde et les taches de tous les glycanes présents dans une molécule de protéine. Par conséquent, l&#39;intensité du signal diminue à mesure que hCGβ est enzymatiquement déglycosylé (voies 1 à 4 voies). Le signal résiduel dans les couloirs 3 et 4 indiquent la présence de motifs glycane, qui sont résistants aux enzymes utilisées. Les glycosidases supplémentaires utilisées dans la piste 4 enlever un peu de résidus de sucre supplémentaire: la migration des protéines est le même, maisune légère réduction de l&#39;intensité de la coloration peut être vu. Fractions de sucre résistant n&#39;étaient pas présents dans toutes les espèces de protéines: certaines bandes n&#39;ont pas été détectés par Emerald Green (absente dans l&#39;image UV, entre parenthèses), indiquant qu&#39;ils ont été largement déglycosylé. Des données additionnelles de soutien à la conclusion que hCGβ est hétérogène glycosylée. La bande inférieure sur la voie 2 (flèche) est faible sur l&#39;image vert émeraude, tandis que la bande supérieure sur la piste 2 est lumineux, ce qui indique que de nombreux groupes de glycanes sont toujours présents. Ces données appuient la conclusion que hCGβ recombinant exprimé dans des cellules de souris contient glycoformes multiples 9. Ces glycoformes différentes sont dues à l&#39;hétérogénéité inhérente de la glycosylation des polypeptides où certains ne reçoivent pas un glycane dans chaque site de consensus et / ou certains glycanes sont étendues tandis que d&#39;autres, même sur la même protéine ne sont pas. <img alt="Figure 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> Figure 1.Modèles de glycosylation typiques pour sécrétée ou glycoprotéines de surface cellulaire. <img alt="Figure 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> Figure 2. Gels SDS-PAGE montrant déglycosylation enzymatique de hCGβ. Le panneau A montre une coloration bleue Coomassie tout panneau B montre les résultats de Pro-Q Emerald 300 pour la visualisation des protéines glycosylées. Numéro de l&#39;échantillon: 1, le contrôle hCGβ; 2, PNGase F digestion; 3, la digestion Mix déglycosylation; 4, ainsi que la digestion Mix déglycosylation exoglycosidases, des échantillons de 5 à 7 sont les contrôles réactifs (O-Glyc, O-glycosidase; GNA, β-N acétylglucosaminidase; Fucosidase Gal / FUC, β1-3 galactosidase, α1-3, 6 galactosidase, et α1-2; Galna, α-N-Acetylglalactosaminidase; Neu, la neuraminidase; PNGase; PNGase F)

<ol>
	<li>Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation:+nature,+distribution,+enzymatic+formation,+and+disease+implications+of+glycopeptide+bonds.">Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.</a> Glycobiology. 12 (4), 43-56 (2002).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+Cellular+Mechanisms+of+Health+and+Disease.">Glycosylation in Cellular Mechanisms of Health and Disease.</a> Cell. 126, 855-867 (2006).</li><li>Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+glycosylation+on+the+biological+function+and+structure+of+human+immunoglobulins.">The impact of glycosylation on the biological function and structure of human immunoglobulins.</a> Annual Review of Immunology. 25, 21-50 (2007).</li><li>Mitra, N., Sinha, S., Ramya, T. N. C., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+oligosaccharides+as+outfitters+for+glycoprotein+folding,+form+and+function.">N-linked oligosaccharides as outfitters for glycoprotein folding, form and function.</a> Trends in Biochemical Sciences. 31, 156-163 (2006).</li><li>Carlsen, R. B., Bahl, O. P., Swaminathan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chorionic+gonadotropin.+Linear+amino+acid+sequence+of+the+beta+subunit.">Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit.</a> Journal of Biological Chemistry. 248 (19), 6810-6812 (1973).</li><li>Tarentino, A. L., Plummer, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deglycosylation+of+Asparagine-linked+Glycans+by+PNGaseF.">Deglycosylation of Asparagine-linked Glycans by PNGaseF.</a> Trends in Glycoscience and Glycotechnology. 5 (23), 163-170 (1993).</li><li>Tretter, V., Altmann, F., Marz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+F+cannot+release+glycans+with+fucose+attached+&#945;1-3+to+the+asparagine-linked+N-acetylglucosamine+residue.">Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached &#945;1-3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199, 647-652 (1991).</li><li>Koutsioulis, D., Landry, D., Guthrie, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+endo-&#945;-N-acetylgalactosaminidases+with+broader+substrate+specificity.">Novel endo-&#945;-N-acetylgalactosaminidases with broader substrate specificity.</a> Glycobiology. 18 (10), 799-805 (2008).</li><li>Thakur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+glycoforms+of+the+intact+alpha+subunit+of+recombinant+human+chorionic+gonadotropin+by+high-resolution+capillary+electrophoresis-mass+spectrometry.">Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 81 (2), 8900-8907 (2009).</li><li>Park, S., Lee, M. R., Shin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+microarrays+as+powerful+tools+in+studies+of+carbohydrate-mediated+biological+processes.">Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.</a> Chemical Communications. 37, 4389-4399 (2008).</li><li>Zachara, N. E., Vosseller, K., Hart, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+proteins+modified+by+O-linked+N-acetylglucosamine.">Detection and analysis of proteins modified by O-linked N-acetylglucosamine.</a> Current Protocols in Molecular Biology. 95, 17-17 (2011).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2008).</li></ol>

Les auteurs sont employés par New England Biolabs, qui produit beaucoup de réactifs utilisés dans cet article.

La méthode décrite ici en utilisant déglycosylation enzymatique et SDS-PAGE peut fournir des informations précieuses sur l&#39;état de glycosylation d&#39;une protéine d&#39;intérêt, tandis que glycane réactifs spécifiques de faciliter l&#39;interprétation des données. Ce protocole est destiné à des études initiales de la glycosylation des protéines et il est particulièrement adapté pour la sécrétion et de glycoprotéines membranaires des cellules de mammifères: les enzymes choisies dans ce cas sera spécifiquement supprimer tous les N-glycanes, et ou N-glycanes plus et les sucres les plus courants l&#39;extension et formant le noyau de O-glycanes. Glycosidases ont l&#39;avantage supplémentaire d&#39;être doux, comparé aux méthodes déglycosylation chimique, en préservant l&#39;intégrité de sucres et de protéines épine dorsale. Pour élucider le taux d&#39;occupation (dont les acides aminés sont glycosylés), l&#39;étendue de la glycosylation, ou pour déterminer la structure fine des glycanes, des techniques plus sophistiquées telles que mspectrométrie de cul, la chromatographie liquide ou RMN sont nécessaires. En raison de sa simplicité, plusieurs étapes de ce protocole peut être ajustée, substitué, et / ou combinés pour s&#39;adapter aux divers besoins expérimentaux. Toutefois, afin d&#39;obtenir des résultats qui peuvent être clairement interprétée, il est important de comprendre ses forces et ses limites. Tout d&#39;abord, la spécificité et la pureté de l&#39;glycosidases sont cruciales: que les enzymes bien caractérisées testé pour être libre de protéases et d&#39;autres activités de contamination doivent être utilisés. Malheureusement, il n&#39;existe pas de définition standard de l&#39;enzyme unité pour les glycosidases, l&#39;utilisateur doit déterminer la substitution appropriée conformément aux spécifications du fabricant. Deuxièmement, un choix judicieux de détection est nécessaire: une réactifs) Coloration des protéines ne sont utiles que si les résultats déglycosylation à un changement important dans la masse moléculaire. Un tel résultat clair comme montré ici n&#39;est pas toujours obtenue. Dans d&#39;autres cas, nous avons vu mi anormalegration après déglycosylation (loin d&#39;être le poids moléculaire prédit, ou la migration encore plus lent). Ce phénomène n&#39;est pas bien comprise, mais on peut dire que tout changement dans la migration est la preuve que la protéine a été déglycosylé. B) la détection de sucre avec des anticorps présente des défis uniques en limitant leur applicabilité. Il a été très difficile de générer générale anti-glycane anticorps, ils sont généralement soulevées contre une cible complexe glycane, ce qui restreint leur utilisation. Par ailleurs, plusieurs anticorps monoclonaux anti-glycane anticorps affichage indésirable réactivité croisée 10. C) Les lectines (protéines ayant une affinité de sucre intrinsèque) sont bien adaptés pour la détection de sucre, plus ils donnent un aperçu sur la structure du glycane. Cependant, pas tous d&#39;entre eux ont une spécificité étroite, et beaucoup ne sont que partiellement caractérisé (ce qui signifie qu&#39;ils pourraient avoir des affinités inconnues). Comme une coloration lectine conséquence positive fournit une indication, pas une preuve, de la présence d&#39;une IGême sucre. d) des kits d&#39;étiquetage de produits chimiques (basée sur l&#39;oxydation au periodate de sucres) sont la méthode de choix pour colorer toutes les glycoprotéines, et donc bien adapté pour suivre déglycosylation. Lors du traitement d&#39;un échantillon inconnu, il est une bonne pratique d&#39;inclure des contrôles glycoprotéine. Fétuine est un N aisément disponibles - et O-glycoprotéine; gonadotrophine chorionique (les deux sous-unités) est également un bon choix. Albumine sérique bovine (BSA) peut être utilisé comme contrôle négatif. Toutefois, il convient de noter que certaines protéines non glycosylées réagissent peu avec le Emerald Pro-Q 300, en particulier lorsqu&#39;il est utilisé à des concentrations élevées. Non-glycosylée standards de poids moléculaire, tels que le marqueur protéique précolorés utilisé dans ce protocole, ont l&#39;avantage d&#39;afficher des bandes nettes. Ce n&#39;est que par hasard est une de ces protéines (80 kDa) réactive à Pro-Q Emerald 300. Par conséquent, l&#39;utilisateur pourrait vouloir lancer une échelle standard glycoprotéine lieu, comme Candy-canne de Invitrogen. Enfin, simple en gel de détection de glycoprotéines nucleocytosolic (qui sont modifiées avec un seul O-GlcNAc) est possible avec l&#39;utilisation d&#39;un anticorps monoclonal, le long de β-N-acétylglucosaminidase pour 11 contrôle de spécificité. La description de cette technique est au-delà des portée de cet article, mais il devrait être mentionné comme glycosidases sont des outils utiles pour l&#39;étude de nombreuses autres glycoprotéines et glycoconjugués présents dans les cellules. Un traitement complet de toutes les formes connues de la glycosylation peuvent être trouvés dans la deuxième édition de la «Essentials of Glycobiologie», disponible gratuitement en ligne à l&#39;étagère de NCBI (ID Bookshelf: NBK1908; PMID: 20301239) 12.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Homme gonadotrophine chorionique β </td><td> Sigma-Aldrich </td><td> C6572 </td><td></td></tr><tr><td> Tubes PCR </td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR Thermocycleur </td><td> Applied Biosystems </td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> P0704 </td><td> Livré avec des tampons </td></tr><tr><td> Mélanger déglycosylation protéines </td><td> New England Biolabs </td><td> P6039 </td><td> Livré avec des tampons </td></tr><tr><td> α-N-Acetylglalactosaminidase </td><td> New England Biolabs </td><td> P0734 </td><td> Livré avec le tampon </td></tr><tr><td> α1-2 Fucosidase </td><td> New England Biolabs </td><td> P0724 </td><td> Livré avec le tampon </td></tr><tr><td> α1-3, 6 Galactosidase </td><td> New England Biolabs </td><td> P0731 </td><td> Livré avec le tampon </td></tr><tr><td> β1-3 Galactosidase </td><td> New England Biolabs </td><td> P0726 </td><td> Livré avec le tampon </td></tr><tr><td> Tampon 10X G7 (500 mM phosphate de sodium) </td><td> New England Biolabs </td><td> --- </td><td> Livré avec PNGaseF ou degl Mix </td></tr><tr><td> Tampon 10X glycoprotéine dénaturation (5% de SDS, 400 mM DTT) </td><td> New England Biolabs </td><td> --- </td><td> Livré avec PNGaseF ou degl Mix </td></tr><tr><td> 10% de NP-40 </td><td> New England Biolabs </td><td> --- </td><td> Livré avec PNGaseF ou degl Mix </td></tr><tr><td> 3X tampon de charge SDS (187,5 mM Tris-HCl pH 6,8, 6% de SDS, glycérol 30%, 0,03% Bromophenol bleu) </td><td> New England Biolabs </td><td> B7703 </td><td> Livré avec 1,25 M de DTT </td></tr><tr><td> ColorPlus précolorés marqueur protéique </td><td> New England Biolabs </td><td> P7709 </td><td></td></tr><tr><td> 10-20% Tris-Glycine Multigel </td><td> Cosmo Bio Co. </td><td> DCB-414893 </td><td></td></tr><tr><td> Unité d&#39;électrophorèse cassette </td><td> Cosmo Bio Co. </td><td> DCB-303111 </td><td></td></tr><tr><td> EPS électrophorèse Alimentation 301 </td><td> GE Healthcare </td><td> 18-1130-01 </td><td></td></tr><tr><td> Pro-Q Kit Emeraude glycoprotéine 300 Stain </td><td> Invitrogen </td><td> P-21857 </td><td></td></tr><tr><td> Brilliant Blue R </td><td> Sigma-Aldrich </td><td> B0149 </td><td></td></tr><tr><td> AlphaImager système HP </td><td> Cellule Biosciences </td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glycosylation, l&#39;addition de sucres liés de façon covalente, est une importante modification post-traductionnelle des protéines qui peuvent affecter de manière significative les processus tels que l&#39;adhésion cellulaire, le trafic moléculaire, dégagement, et 1-4 transduction du signal. Chez les eucaryotes, les modifications de la glycosylation les plus courantes dans la voie de sécrétion sont des ajouts au niveau des résidus asparagine consensus (N-liés); ou à résidus serine ou thréonine (O-lié) (figure 1). Initiation de la N-glycanes de synthèse est très conservée chez les eucaryotes, tandis que les produits finaux peuvent varier considérablement entre les différentes espèces, les tissus ou les protéines. Certains glycanes sont pas modifiées («high mannose N-glycanes») ou sont traitées ultérieurement dans le Golgi (&quot;complexe N-glycanes»). Une plus grande diversité se trouve pour O-glycanes, qui commencent par une commune N-acétylgalactosamine (GalNAc) de résidus dans les cellules animales, mais diffèrent dans les organismes inférieurs 1. ent &quot;&gt; L&#39;analyse détaillée de la glycosylation des protéines est un domaine en soi et nécessite d&#39;importantes ressources et l&#39;expertise pour exécuter correctement. Cependant une variété d&#39;enzymes disponibles que les sucres supprimer (glycosidases) rend possible d&#39;avoir une idée générale de l&#39;état de glycosylation des . protéine dans un laboratoire standard ici, nous illustrons l&#39;utilisation de glycosidases pour l&#39;analyse d&#39;une glycoprotéine modèle:. gonadotrophine chorionique humaine recombinante bêta (hCGβ), qui porte deux N-glycanes et quatre O-glycanes 5 La technique ne nécessite que simples l&#39;instrumentation et des consommables typique, et il peut être facilement adapté à l&#39;analyse des échantillons glycoprotéine multiples. Plusieurs enzymes peuvent être utilisées en parallèle pour étudier une glycoprotéine. PNGase F est capable d&#39;éliminer presque tous les types de N-glycanes 6,7. Pour O-glycanes, il n&#39;ya pas d&#39;enzymes disponibles qui peuvent cliver un oligosaccharide intact depuis èmesquelette protéique e. Au lieu de cela, O-glycanes sont garnis par des exoglycosidases à une courte carotte, qui est alors facilement enlevé par O-glycosidase. Le Grand Mix déglycosylation protéine contient PNGase F, O-glycosidase, la neuraminidase (sialidase), β1-4 galactosidase et β-N-acétylglucosaminidase. Il est utilisé pour retirer simultanément N-glycanes et certains glycanes O-8. Enfin, le Mix déglycosylation a été complétée avec un mélange de exoglycosidases autres (α-N-acétylgalactosaminidase, α1-2 Fucosidase, α1-3, 6 galactosidase, et β1-3 galactosidase), qui aident à éliminer les monosaccharides contraire résistants qui pourraient être présents dans certains O-glycanes. SDS-PAGE/Coomasie bleu est utilisé pour visualiser les différences en matière de migration des protéines avant et après traitement glycosidase. En outre, un sucre spécifique méthode de coloration, Proq Emeraude-300, montre que le signal diminue glycanes sont successively enlevé. Ce protocole est conçu pour l&#39;analyse de petites quantités de glycoprotéines (0,5 à 2 ug), bien que déglycosylation enzymatique peut être étendu pour accueillir de grandes quantités de protéines que nécessaire.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Utiliser glycosidases spécifiques pour éliminer les sucres de glycoprotéines suivi par SDS-PAGE est une méthode utile pour détecter des modifications glycane sur des échantillons de protéines et est un bon choix pour des études glycobiologie initiale. Les changements suivants déglycosylation peuvent être détectés comme des changements dans la mobilité de gel ou par coloration avec glycane réactifs sensibles.

Identification et caractérisation des glycosylation des protéines spécifiques en utilisant des endo-et exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

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Biology

36.4K vues.  New England Biolabs. Utiliser glycosidases spécifiques pour éliminer les sucres de glycoprotéines suivi par SDS-PAGE est une méthode utile pour détecter des modifications glycane sur des échantillons de protéines et est un bon choix pour des études glycobiologie initiale. Les changements suivants déglycosylation peuvent être détectés comme des changements dans la mobilité de gel ou par coloration avec glycane réactifs sensibles.

Vidéo: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identification des complexes protéiques avec la protéomique quantitative chez S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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Biologie

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utiliser le système de fractionnement GELFREE 8100 pour les Poids Moléculaire à base de fractionnement avec récupération de la phase liquide

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identification des partenaires protéine interagissant Utilisation Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Microarray Anticorps chimiquement bloqué pour multiplexée à haut débit de profilage de glycosylation des protéines spécifiques dans les échantillons complexes

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Méthode pour l&#39;isolement et l&#39;identification des ARNm, les microARN et composants protéiques des complexes ribonucléoprotéiques partir d&#39;extraits cellulaires utilisant RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identification des complexes protéiques dans<em&gt; Escherichia coli</em&gt; En utilisant séquentielle Purification par affinité peptide en combinaison avec la spectrométrie de masse en tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Production, de conditionnement, et la caractérisation des cellules invasives Espèces protéine DISC1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing pour l&#39;identification microbienne et Caractérisation

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Isolement des régions génomiques spécifiques et l&#39;identification des molécules associées par enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.