Cette étude a été soutenue par un prix d&#39;excellence du ministère américain des Anciens Combattants, et une subvention du NIH R01CA138528 à RW.

1 Préparation de constructions d&#39;expression <ol><li> Avant de commencer le protocole, PRMT clones dans des vecteurs d&#39;expression de mammifère avec une étiquette d&#39;épitope N-terminal (Myc ou HA), et le substrat dans le cadre avec la glutathion-S-transférase (GST) pour l&#39;expression de protéine à haut niveau et la purification à partir de cellules bactériennes lysats, en utilisant des techniques classiques de biologie moléculaire. </li></ol> 2 Purification de PRMT <ol><li> Utilisez des boîtes de 100 mm de culture pour maintenir les cellules épithéliales bronchiques humaines (Beas2B). Passage des cellules tous les 3 jours et ne les laissez pas se développer jusqu&#39;à confluence. </li><li> Le jour avant la transfection, semences 7,5 x 10 5 cellules dans 10 ml de milieu de culture dans une boîte de culture de 100 mm. Agiter pour disperser uniformément les cellules et incuber à 37 ° C dans un incubateur à CO2 humidifié à 5%. </li><li> Le lendemain, aspirer les médias et ajouter 5 ml de milieu de culture sans sérum et antibiotiques. Agiter et aspirer le support. Répétez cette étape unplus de temps, et, enfin, ajouter 5 ml d&#39;/ milieu sans antibiotique de sérum et de procéder à la transfection. </li><li> Diluer 6 pg d&#39;ADN de plasmide dans 750 ul de milieu sans sérum dans un tube en plastique de 1,5 ml de centrifugation stérile. </li><li> Dans un autre tube à centrifuger en plastique de 1,5 ml, diluer 30 pi de réactif de transfection dans 750 ul de milieu sans sérum. </li><li> Transférer la solution de réactif de transfection dilué (étape 2.5) dans le tube contenant l&#39;ADN dilué (étape 2.4). Mélanger doucement par aspiration et à plusieurs reprises. </li><li> Incuber le mélange de transfection à la température ambiante pendant 15 min. </li><li> Après 15 min, la pipette le mélange de transfection doucement sur ​​les cellules, agiter pour mélanger uniformément, et incuber à 37 ° C dans un incubateur humidifié CO 2 de 5%. </li><li> Après 3 heures, aspirer le milieu contenant les mélanges de transfection des cellules. Ajouter du milieu frais de culture de cellules additionné de sérum et d&#39;antibiotiques. </li><li> Incuber à 37 ° C dans un CO humidifié à 5% 2incubateur pendant 48 heures. </li><li> Après 48 h, de lyser les cellules dans 1 ml de tampon de lyse (1x solution saline tamponnée phosphate, 1% Nonidet P-40, le désoxycholate de sodium à 0,5%, 0,1% de dodécylsulfate de sodium, et 1 ug / ml phenlymethyl sulfonyl fluorure). Recueillir les lysats dans un tube de centrifugeuse en matière plastique avec un dispositif de levage de pile et incuber les lysats sur de la glace pendant 30 min. </li><li> Centrifuger les lysats à 15 000 xg pendant 10 min sur une centrifugeuse de paillasse pour éliminer les lysats à partir des débris cellulaires. [* Il est fortement recommandé de confirmer l&#39;expression de HA-PRMT1 par immunoblot d&#39;une aliquote de lysats avec des anticorps anti-HA, avant de procéder à la purification des enzymes pour le dosage de la méthylation.] </li><li> Pour la purification de PRMT, incuber 3 mg de lysats de cellules avec 30 pl de suspension de 50:50 anti-HA matrice d&#39;affinité dans une solution saline tamponnée au phosphate (PBS), et faire tourner pendant la nuit à 4 ° C dans un agitateur de tubes. </li><li> Le jour suivant, tourner les tubes à 14000 xg dans une centrifugeuse de paillasse pendant 2 min à collecterles billes. Jeter le surnageant et laver les billes 3 fois avec 1 ml de tampon RIPA [20 mM Tris (pH 8,0), NaCl 150 mM, EDTA 5 mM et 1% de Triton-X-100] à chaque lavage suivie d&#39;une centrifugation à 14 000 xg pendant 2 min. Après le troisième lavage, laver les billes une fois avec 100 pl de tampon de méthylation (50 mM Tris pH 8,5, 20 mM KCl, 10 mM de MgCl2, 1 mM β-mercaptoéthanol, 100 mM de saccharose). Après la centrifugation, éliminer le surnageant. Les perles contenant des PRMT immobilisées sont prêts à être utilisée dans le dosage de la méthylation. [* Il est fortement recommandé d&#39;utiliser immédiatement les PRMT immobilisés dans le test de méthylation.] </li></ol> 3 Purification de substrat <ol><li> Inoculer une colonie unique de GST-G3BP1 BL21 transformé dans 5 ml de milieu Luria-Bertani (LB) contenant 100 ug / ml d&#39;ampicilline. Cultiver les cultures pendant une nuit à 37 ° C sous agitation à 250 tours par minute. </li><li> Le lendemain, transférer la culture de la nuit à 50 ml de milieu LB frais et suiteinue de croître à une DO600 de 0,5-0,7 (Il faut environ 2 heures). </li><li> Lorsque la DO600 atteigne 0,5 à 0,7, ajouter isopropyl-β-D-thiogalactoside (IPTG) à une concentration finale de 1 mM et de continuer la culture pendant encore 4 heures à 37 ° C sous agitation à 250 tours par minute. </li><li> Récolter les cellules par centrifugation à 1 500 g pendant 10 min, et traiter le culot pour la purification de la TPS ou conserver à -80 ° C. </li><li> Re-suspendre le culot bactérien dans 5 ml de tampon de lyse (PBS 1x avec des inhibiteurs de la protéase, de 100 pg / ml de lysozyme et 0,1% de Triton-X-100) et on incube les lysats à 37 ° C pendant 30 min. A cette époque, les lysats apparaîtront trouble et visqueuse due à la libération de l&#39;ADN génomique bactérienne. </li><li> Cisailler l&#39;ADN génomique par traitement aux ultrasons (10 impulsions d&#39;une seconde chacune avec une amplitude de 30%). </li><li> Effacer les lysats par centrifugation à 15 000 g pendant 10 min et transférer le surnageant clair à un tube de 15 ml. Si le surnageant n&#39;est pas clair répéter la centrifuprocessus gation un plus de temps. </li><li> Pendant ce temps, laver les billes de TPS-sépharose deux fois avec du PBS froid et faire une suspension à 50:50 de GST-sépharose dans du PBS. Ajouter 200 ul de la suspension de GST-sépharose au lysat de cellules bactériennes dégagée et tourner à 4 ° C pendant 1 heure. </li><li> Après 1 heure, centrifuger les tubes à 500 g pendant 3 min pour recueillir les perles. Lavez les perles 3x avec le froid de la glace PBS. </li><li> Après le lavage final, remettre en suspension les billes dans 100 pl de tampon d&#39;élution (50 mM Tris pH 8,0, 150 mM de NaCl, 30 mM de glutathion) et tourner à 4 ° C pendant 10 min. </li><li> Centrifuger les tubes à 15 000 x g pendant 2 min et transférer le surnageant contenant la protéine purifiée dans un autre tube à centrifuger en plastique. Effectuer gel de polyacrylamide SDS (SDS-PAGE) d&#39;analyse de 5 pi de la protéine purifiée avec des quantités connues d&#39;albumine de sérum bovin (BSA) afin d&#39;estimer la concentration de la protéine purifiée. </li></ol> 4. test de Méthylation REMARQUE: Effectuez les étapes suivantes dans une zone séparée désigné pour le travail de radio-isotopes et en conformité avec les protocoles de l&#39;établissement sur la réalisation d&#39;expériences avec des isotopes radioactifs. <ol><li> Ajouter 2 ug de GST-substrat purifiée et un mélange maître contenant un tampon de méthylation 1x (50 mM Tris pH 8,5, 20 mM de KCl, 10 mM de MgCl2, 1 mM β-mercaptoéthanol, saccharose 100 mM), 1 uCi de S-adenosyl- L-[méthyl-3H] methionine, et de l&#39;eau jusqu&#39;à un volume final de 10 ul aux PRMT immobilisées de l&#39;étape (2.13) et incuber la réaction à 30 ° C pendant 1 heure. </li><li> Après 1 h, stopper la réaction par addition de 10 pi de tampon d&#39;échantillon SDS 2x. Après dénaturation des échantillons à 95 ° C pendant 10 minutes, séparer les échantillons sur un gel SDS-PAGE. </li><li> Par la suite, le transfert des protéines séparées dans le gel SDS sur une membrane de nitrocellulose en utilisant un transfert électrophorétique semi-sec. </li><li> Après le transfert, couvrir la membrane avec unutofluor (une image de intensificateur autoradiographique), et bercer doucement pendant 2 h à température ambiante. Après 2 h, verser le autofluor et le conserver pour un usage ultérieur. [* Etiqueter le tube de matière radioactive] </li><li> Air rapidement sécher la membrane sur un papier filtre, l&#39;enfermer dans un sac en plastique et d&#39;exposer la membrane à un film X-ray à -20 ° C. Durée d&#39;exposition suffisante varie entre 5-30 jours. </li></ol> 5 Confirmation d&#39;expression et de l&#39;égalité de PRMT Chargement de substrat <ol><li> Après la réalisation de l&#39;exposition suffisantes, transférer la membrane à une boîte en plastique contenant un tampon de blocage [solution saline tamponnée au Tris avec du Tween-20 (TBST + 3% de lait écrémé)] et balancer doucement pendant 1 heure. </li><li> Jetez le tampon de blocage en tant que déchets radioactifs. Incuber la membrane avec l&#39;anticorps anti-HA (1: 1000 dilution) dans du TBST pendant une nuit à 4 ° C, avec des doux balancement. </li><li> Laver la membrane avec du TBST 3x et incuber la membrane avec un anticorps secondaire anti-souris marqué à la HRP(1: 5000 dilution) dans du TBST à température ambiante pendant 1 h avec doux balancement. </li><li> Après 1 heure, laver la membrane avec du TBST 3x, et effectuer la détection de chimiluminescence de protéines avec un substrat HRP. </li><li> Après détection des PRMT HA-tags, incuber la membrane avec une solution de coloration Ponseau-S pendant 5 min à température ambiante. Laver rapidement les 3x membrane avec de l&#39;eau pour détecter les substrats de la TPS-étiqueté. Numériser l&#39;image. </li></ol>

<ol>
	<li>Paik, W. K., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+methylase+I.+Purification+and+properties+of+the+enzyme.">Protein methylase I. Purification and properties of the enzyme.</a> J Biol Chem. 243, 2108-2114 (1968).</li><li>Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., Herschman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+immediate-early+TIS21+protein+and+the+leukemia-associated+BTG1+protein+interact+with+a+protein-arginine+N-methyltransferase.">The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase.</a> J Biol Chem. 271, 15034-15044 (1996).</li><li>Boffa, L. C., Karn, J., Vidali, G., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+NG,+NG,-dimethylarginine+in+nuclear+protein+fractions.">Distribution of NG, NG,-dimethylarginine in nuclear protein fractions.</a> Biochem Biophys Res Commun. 74, 969-976 (1977).</li><li>Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+at+a+glance.">Arginine methylation at a glance.</a> J Cell Sci. 120, 4243-4246 (2007).</li><li>Lee, Y. H., Stallcup, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+protein+arginine+methylation+of+nonhistone+proteins+in+transcriptional+regulation.">Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.</a> Mol Endocrinol. 23, 425-433 (2009).</li><li>Bedford, M. T., Clarke, S. 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+arginine+methylation+in+mammals:+who,+what,+and+why.">Protein arginine methylation in mammals: who, what, and why.</a> Mol Cell. 33, 1-13 (2009).</li><li>Bikkavilli, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled3+is+a+novel+arginine+methyl+transferase+substrate.">Dishevelled3 is a novel arginine methyl transferase substrate.</a> Scientific reports. 2, 805 (2012).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+of+G3BP1+in+response+to+Wnt3a+regulates+{beta}-catenin+mRNA.">Arginine methylation of G3BP1 in response to Wnt3a regulates {beta}-catenin mRNA.</a> J Cell Sci. 124, 2310-2320 (2011).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt3a-stimulated+LRP6+phosphorylation+is+dependent+upon+arginine+methylation+of+G3BP2.">Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.</a> J Cell Sci. , (2012).</li><li>Tini, M., Naeem, H., Torchia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+arginine+methylation+in+transcription.">Biochemical analysis of arginine methylation in transcription.</a> Methods Mol Biol. 523, 235-247 (2009).</li><li>Lee, J., Cheng, D., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+protein+methylation.">Techniques in protein methylation.</a> Methods Mol Biol. 284, 195-208 (2004).</li><li>Cheng, D., Vemulapalli, V., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+applied+to+the+study+of+protein+arginine+methylation.">Methods applied to the study of protein arginine methylation.</a> Methods Enzymol. 512, 71-92 (2012).</li><li>Zurita-Lopez, C. I., Sandberg, T., Kelly, R., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+protein+arginine+methyltransferase+7+(PRMT7)+is+a+type+III+enzyme+forming+omega-NG-monomethylated+arginine+residues.">Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming omega-NG-monomethylated arginine residues.</a> J Biol Chem. 287, 7859-7870 (2012).</li></ol>

Les auteurs n&#39;ont rien à divulguer.

Le protocole décrit ici est couramment utilisée pour établir le statut de méthylation des substrats de PRMT identifiés. Ce test robuste et cohérente sera également apporter la preuve définitive de la spécificité de PRMT pour leurs substrats. Les éléments clés pour la réussite de ce test sont les suivants: 1 Expression de PRMT dans les cellules de mammifères, 2 Activité de PRMT purifiées, 3 Expression et purification de substrat, et 4 transfert occidentale complète des protéines de la membrane de nitro...

Méthylation des protéines a été décrite pour la première en 1968 1. Ce n&#39;est que le premier clonage d&#39;PRMT1 en 1996, que les chercheurs ont commencé à comprendre l&#39;importance de cette modification post-traductionnelle 2. Fait intéressant, environ 2% des résidus arginine dans les protéines d&#39;extraits nucléaires sont méthylés 3, ce qui indique l&#39;abondance de cette modification. L&#39;arginine est un acide aminé chargé positivement avec une chaîne latérale de base et la teneur en azote / s dans les chaînes latérales de l&#39;arginine peut être modifié après la traduction par l&#39;intermédiaire de l&#39;addition d&#39;un groupe méthyle, un processus connu comme l&#39;arginine méthylation 6.4. Arginine méthylation est catalysée par une classe d&#39;enzymes à savoir. Protéine transférases, méthyle arginine (PRMT). Arginines peuvent être soit monométhylées ou diméthylées et celui-ci peut être soit symétrique ou asymétrique selon le type de PRMT catalysant le procédé 4-6. Les protéines qui présentent arginineriches motifs de e-glycine sont des cibles potentielles pour la catalyse de PRMT médiation. méthylation de PRMT médiée par des substrats a été montré pour moduler l&#39;interaction protéine-protéine, une interaction protéine-acide nucléique, la fonction des protéines, l&#39;expression des gènes, et / ou de signalisation cellulaire, qui sont critiques pour l&#39;homéostasie cellulaire normale 7-9. Afin de comprendre le rôle biologique de la protéine méthylation de l&#39;arginine, les dosages précis, efficaces et reproductibles sont nécessaires pour établir le statut de méthylation des substrats de PRMT identifiés. En test de méthylation in vitro, qui évalue les capacités de PRMT purifiées à catalyser la méthylation de leurs substrats, est un test bien accepté pour étudier la méthylation de l&#39;arginine 10-12. Le succès global de ce dosage dépend largement de l&#39;activité des PRMT purifiés. PRMT peuvent être exprimées et purifiées à partir de bactéries ou de cellules de mammifères, 10,11. Ce dosage in vitro de la méthylation, comme detailed dans la partie protocole, est basée sur un procédé décrit à l&#39;origine par Tini et collègues 10. Dans ce protocole, on montre en détail les étapes intervenant dans l&#39;expression et la purification de PRMT1 dans des cellules de mammifères. La capacité de la PRMT1 purifiée pour catalyser la méthylation de Ras-GTPase activating protein protéine de liaison 1 (G3BP1), un substrat connu PRMT 8, a ensuite été évaluée en présence de S-adénosyl-L-[méthyl-3H] methionine en tant que donneur de méthyle. En utilisant ce dosage, on peut définir de manière fiable les capacités des PRMT à nouveau le méthylate ou substrats connus, ce qui est une étape primordiale dans l&#39;étude de la protéine méthylation de l&#39;arginine.

<table border="0" cellpadding="0" cellspacing="0" width="673">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">S-adenosyl-L-[methyl-3H] methionine</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ecoscint ultra&#160;</td>
			<td>National Diagnostics</td>
			<td>LS-270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bottle top dispenser</td>
			<td>National Diagnostics</td>
			<td>LS-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoFluor</td>
			<td>National Diagnostics</td>
			<td>LS-315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 ml Scintillation Vials</td>
			<td>National Diagnostics</td>
			<td>SKU:SVC-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GST-sepharose</td>
			<td style="width:136px;">GE Life Sciences</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-Glutathione Reduced</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;">G4251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Lipofectamine</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:111px;"></td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Beas2B</td>
			<td style="width:136px;">ATCC</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Optimem</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NP-40</td>
			<td style="width:136px;">US-Biologicals</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">SDS</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sodium deoxycholate</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PMSF</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">anti-HA affinity matrix</td>
			<td style="width:136px;">Roche</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Tris</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nacl</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Bio-rad gel doc</td>
			<td style="width:136px;">Bio Rad</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">anti-HA antibody</td>
			<td style="width:136px;">roche</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Ponseau S</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro methylation assay to study protein arginine methylation

Protéine méthylation de l&#39;arginine est l&#39;une des modifications post-traductionnelles les plus abondants dans le noyau. Protéine méthylation de l&#39;arginine peut être identifié et / ou déterminé par l&#39;intermédiaire d&#39;approches de protéomique, et / ou immunotransfert avec des anticorps spécifiques méthyl-arginine. Cependant, ces techniques peuvent parfois être trompeuses et fournissent souvent des résultats faussement positifs. Plus important encore, ces techniques ne peuvent pas fournir une preuve directe à l&#39;appui de la spécificité de substrat de PRMT. Des essais in vitro de méthylation, d&#39;autre part, sont des dosages biochimiques utiles, qui sont sensibles, et révèlent systématiquement si les protéines identifiées sont en effet des substrats PRMT. Un dosage typique de méthylation in vitro, comprenant PRMT actives purifiées substrat purifié, et un radio-isotope marqué donneur de méthyle (S-adénosyl-L-[méthyl-3H] methionine). Nous décrivons ici un protocole étape par étape pour isoler PRMT1 activité catalytique, un membre de la famille de PRMT exprimé de façon ubiquitaire. Le moiThyl activités de transferase de la PRMT1 purifiés ont ensuite été testés sur Ras-GTPase activant la protéine de liaison 1 (G3BP1) protéine, un substrat de PRMT connu, en présence de S-adénosyl-L-[méthyl-3H] methionine en tant que donneur de méthyle. Ce protocole peut être utilisé non seulement pour l&#39;établissement de l&#39;état de méthylation de nouveaux substrats de PRMT1 physiologiques, mais également pour la compréhension du mécanisme de base de la méthylation de l&#39;arginine protéine.

<html> Les capacités de HA-PRMT1 de méthylate TPS-G3BP1 ont été déterminées par un test in vitro de méthylation. PRMT1 étiquetée HA-purifié à partir de lysats de cellules Beas2B a été utilisé dans une réaction de méthylation contenant GST-G3BP1 et 1 uCi de S-adénosyl-L-[méthyl-3H] methionine, comme donneur de méthyle. PRMT1 pourrait efficacement catalyser la méthylation de TPS-G3BP1 (figure 1, partie supérieure). réactions de méthylation à l&#39;aide de listes ...

Watch this Scientific Journal Video about In vitro Methylation Assay to Study Protein Arginine Methylation at JoVE.com

In vitro Methylation Assay to Study Protein Arginine Methylation

Protéine méthylation de l&#39;arginine, catalysée par une classe d&#39;enzymes à savoir. Protéine arginine méthyl transférases (en PRMT), est le processus d&#39;addition enzymatique de groupe (s) méthyle arginines à l&#39;intérieur des protéines. Le test de méthylation in vitro est l&#39;outil le plus fiable pour évaluer l&#39;état de méthylation de substrats connus ou nouveaux PRMT.

In vitro test de Méthylation à étudier des protéines méthylation de l&#39;arginine

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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Research

JoVE Journal

Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

15.1K vues.  University of Illinois at Chicago.  Protéine méthylation de l'arginine, catalysée par une classe d'enzymes à savoir. Protéine arginine méthyl transférases (en PRMT), est le processus d'addition enzymatique de groupe (s) méthyle arginines à l'intérieur des protéines. Le test de méthylation in vitro est l'outil le plus fiable pour évaluer l'état de méthylation de substrats connus ou nouveaux PRMT. 

Vidéo: In vitro Methylation Assay to Study Protein Arginine Methylation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Détermination de méthylation de l&#39;ADN des gènes soumis à empreinte dans les Arabidopsis Endosperme

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Essai de superposition des protéines membranaires: Un protocole pour tester l&#39;interaction entre les protéines solubles et insolubles In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Méthylation de l&#39;ADN: bisulfite de modification et d&#39;analyse

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un essai en phase liquide d&#39;affinité de capture utilisant des billes magnétiques pour étudier interaction protéine-protéine: Le poliovirus Exemple-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

Application d&#39;un<em&gt; In vitro</em&gt; DNA Assay Protection de bien visualiser les propriétés de médiation du stress de la protéine Dps

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Des cellules endothéliales Tube Formation test pour la<em&gt; In Vitro</em&gt; Etude de l&#39;angiogenèse

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Enhanced réduit Représentation bisulfite séquençage pour l&#39;évaluation de méthylation de l&#39;ADN à la résolution de paires de base

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Corréler spécifique du gène ADN changements méthylation avec l&#39;expression et l&#39;activité transcriptionnelle des astrocytes<em&gt; KCNJ10</em&gt; (Kir4.1)