Questo studio è stato sostenuto da un Merit Award dalla US Department of Veterans Affairs, e un NIH concedere R01CA138528 a RW.

1 Preparazione di costrutti di espressione <ol><li> Prima di iniziare il protocollo, PRMTs clone in vettori di espressione di mammifero con un epitopo tag N-terminale (Myc o HA), e il substrato in-frame con glutatione-S-transferasi (GST) per l&#39;espressione di proteine ​​di alto livello e la purificazione da cellule batteriche lisati, utilizzando tecniche di biologia molecolare standard. </li></ol> 2 Purificazione di PRMTs <ol><li> Usare piatti 100 mm di coltura per mantenere le cellule umane epiteliali bronchiali (Beas2B). Passage le cellule ogni 3 giorni e non lasciarli crescere a confluenza. </li><li> Il giorno prima della trasfezione, seminare 7.5 x 10 5 cellule in 10 ml di terreno di coltura in un piatto di cultura a 100 mm. Agitare per disperdere uniformemente le cellule, e incubare a 37 ° C in un incubatore umidificato CO 2 5%. </li><li> Il giorno successivo, aspirare i media e aggiungere 5 ml di coltura senza siero e antibiotici. Agitare e aspirare il terreno. Ripetere questo passo sipiù tempo, e, infine, aggiungere 5 ml di siero / terreno privo di antibiotici e procedere alla trasfezione. </li><li> Diluire 6 mg di DNA plasmidico in 750 ml di mezzo privo di siero in una sterile da 1,5 ml di tubo di plastica da centrifuga. </li><li> In un altro 1,5 ml tubo da centrifuga di plastica, diluire 30 ml di reagente di trasfezione in 750 ml di mezzo privo di siero. </li><li> Trasferire la soluzione reagente di trasfezione diluito (punto 2.5) alla provetta contenente DNA diluito (punto 2.4). Mescolare delicatamente pipettando su e giù parecchie volte. </li><li> Incubare la miscela di trasfezione a temperatura ambiente per 15 min. </li><li> Dopo 15 minuti, pipettare la miscela di trasfezione delicatamente sulle celle, agitare per mescolare in modo uniforme, e incubare a 37 ° C in un incubatore umidificato CO 2 5%. </li><li> Dopo 3 ore, aspirare il terreno contenente miscele di trasfezione delle cellule. Aggiungere fresco terreno di coltura cellulare integrato con siero e antibiotici. </li><li> Incubare a 37 ° C in un umidificata al 5% di CO 2incubatore per 48 ore. </li><li> Dopo 48 ore, lisare le cellule in 1 ml di tampone di lisi (1x tampone fosfato salino, 1% Nonidet P-40, 0,5% desossicolato di sodio, 0,1% di sodio dodecil solfato, e 1 mg / ml phenlymethyl sulfonil fluoruro). Raccogliere i lisati in un tubo da centrifuga di plastica con un sollevatore cellulare e incubare i lisati in ghiaccio per 30 min. </li><li> Centrifugare i lisati a 15.000 xg per 10 minuti su una centrifuga da banco per cancellare i lisati da detriti cellulari. [* Si consiglia vivamente di confermare l&#39;espressione di HA-PRMT1 tramite immunoblotting di una aliquota dei lisati con anticorpi anti-HA, prima di procedere alla purificazione degli enzimi per l&#39;analisi di metilazione.] </li><li> Per la purificazione di PRMTs, incubare 3 mg di lisati cellulari con 30 ml di sospensione 50:50 di anti-HA matrice di affinità in tampone fosfato salino (PBS), e ruotare notte a 4 ° C in un rotatore tubo. </li><li> Il giorno dopo, girare le provette a 14000 g in una centrifuga da banco per 2 minuti per raccoglierele perline. Eliminare il surnatante e lavare le perline 3x con 1 ml di tampone RIPA [20 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA e 1% Triton-X-100] in ogni lavaggio seguito da centrifugazione a 14000 g per 2 min. Dopo il terzo lavaggio, lavare le perle di una volta con 100 ml di buffer di metilazione (50 mM Tris pH 8.5, 20 mM KCl, 10 mM MgCl 2, 1 mM β-mercaptoetanolo, saccarosio 100 mm). Dopo la centrifugazione, rimuovere il supernatante. Le perle contenenti i PRMTs immobilizzati sono pronti per essere utilizzati nel test di metilazione. [* Si consiglia vivamente di utilizzare i PRMTs immobilizzati immediatamente nel test di metilazione.] </li></ol> 3. Purificazione di substrato <ol><li> Inoculare una singola colonia di GST-G3BP1 trasformato BL21 in 5 ml di Luria-Bertani (LB) medium contenenti 100 mg / ml di ampicillina. Far crescere le culture per una notte a 37 ° C con agitazione a 250 rpm. </li><li> Il giorno dopo, trasferire la cultura durante la notte a 50 ml di terreno LB fresco e continue a crescere ad un OD 600 di 0,5-0,7 (Ci vogliono circa 2 ore). </li><li> Quando il diametro esterno 600 raggiunge 0,5-0,7, aggiungere isopropil-β-D-thiogalactoside (IPTG) ad una concentrazione finale di 1 mM e continuare a cultura per ulteriori 4 ore a 37 ° C con agitazione a 250 rpm. </li><li> Raccogliere le cellule per centrifugazione a 1500 xg per 10 minuti, ed elaborare il pellet per la purificazione GST o conservare a -80 ° C. </li><li> Risospendere il pellet batterico in 5 ml di tampone di lisi (PBS 1x con inibitori della proteasi, 100 ug / ml di lisozima e 0,1% Triton-X-100) e incubare i lisati a 37 ° C per 30 min. In questo momento i lisati appariranno torbido e viscoso a causa del rilascio di DNA genomico batterico. </li><li> Shear il DNA genomico mediante sonicazione (10 impulsi di 1 secondo ciascuno con un&#39;ampiezza di 30%). </li><li> Cancellare i lisati di centrifugazione a 15.000 xg per 10 minuti e trasferire il surnatante limpido in una provetta da 15 ml. Se il surnatante è chiaro non ripetere l&#39;centrifugogazione processo ancora una volta. </li><li> Nel frattempo, lavare le perle GST-sefarosio due volte con PBS freddo ghiaccio e fare una sospensione di 50:50 GST-sefarosio in PBS. Aggiungere 200 microlitri della sospensione GST-sefarosio al lisato cellulare batterica eliminato e ruotare a 4 ° C per 1 ora. </li><li> Dopo 1 ora, centrifugare le provette a 500 xg per 3 minuti per raccogliere le perline. Lavare le perline 3x con ghiaccio freddo PBS. </li><li> Dopo l&#39;ultimo lavaggio, risospendere le sfere in 100 ml di tampone di eluizione (50 mM Tris pH 8,0, 150 mM NaCl, 30 mM glutatione) e ruotare a 4 ° C per 10 min. </li><li> Centrifugare le provette a 15.000 xg per 2 minuti e trasferire il surnatante contenente la proteina purificata in un&#39;altra provetta da centrifuga di plastica. Effettuare SDS elettroforesi su gel di poliacrilammide (SDS-PAGE) analisi su 5 microlitri della proteina purificata insieme con quantità note di albumina di siero bovino (BSA) per stimare la concentrazione della proteina purificata. </li></ol> 4. Metilazione Assay NOTA: Effettuare operazioni seguenti in una zona separata designata per il lavoro radioisotopo e in conformità con i protocolli delle istituzioni su come eseguire gli esperimenti con isotopi radioattivi. <ol><li> Aggiungere 2 mg di purificato GST-substrato e una master mix contenente tampone metilazione 1x (50 mM Tris pH 8.5, 20 mM KCl, 10 mM MgCl 2, 1 mM β-mercaptoetanolo, saccarosio 100 mm), 1 μCi di S-adenosyl- L- [metil-3H] metionina e acqua ad un volume finale di 10 pl alle PRMTs immobilizzati (passo 2.13) e incubare la reazione a 30 ° C per 1 ora. </li><li> Dopo 1 ora, fermare la reazione aggiungendo 10 ml di tampone campione 2x SDS. Dopo denaturazione dei campioni a 95 ° C per 10 min, separare i campioni su un gel SDS-PAGE. </li><li> Più tardi, trasferire le proteine ​​separate dal gel SDS su membrana di nitrocellulosa mediante un trasferimento elettroforetico semi-secco. </li><li> Dopo il trasferimento, coprire la membrana con unutofluor (un intensificatore di immagine autoradiografica), e delicatamente roccia per 2 ore a temperatura ambiente. Dopo 2 ore, versare fuori il autofluor e conservare per un uso successivo. [* Etichettare la provetta come materiale radioattivo] </li><li> Aria rapidamente asciugare la membrana su una carta da filtro, sigillarlo in un sacchetto di plastica e di esporre la membrana di una pellicola a raggi X a -20 ° C. Tempo di esposizione sufficiente varia tra 5-30 giorni. </li></ol> 5 Conferma di PRMT espressione e Pari carico di substrato <ol><li> Dopo aver raggiunto le esposizioni sufficienti, trasferire la membrana di una scatola di plastica contenente tampone bloccante [salina tamponata con Tris con Tween-20 (TBST + latte scremato 3%)] e scuoterla delicatamente per 1 ora. </li><li> Scartare il tampone bloccante come rifiuti radioattivi. Incubare la membrana con anticorpi anti-HA (1: 1.000 diluizione) in TBST notte a 4 ° C, con dolce dondolio. </li><li> Lavare la membrana con TBST 3x e incubare la membrana con l&#39;anticorpo secondario HRP-marcato anti-topo(1: 5.000 diluizione) in TBST a temperatura ambiente per 1 ora con dolce dondolio. </li><li> Dopo 1 ora, lavare la membrana con TBST 3x, ed eseguire il rilevamento chemiluminescenza di proteine ​​con un substrato HRP. </li><li> Dopo la rilevazione dei PRMTs HA-tag, incubare la membrana con soluzione colorante Ponseau-S per 5 minuti a temperatura ambiente. Lavare rapidamente le 3x membrana con acqua per rilevare i substrati GST-tagged. Eseguire la scansione dell&#39;immagine. </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>

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Il protocollo qui descritto è abitualmente utilizzato per stabilire lo stato di metilazione dei substrati PRMT identificati. Questo test robusto e coerente fornirà anche prove definitive sulla specificità del PRMTs per i loro substrati. I componenti chiave per il successo di questo test sono: 1 Espressione di PRMTs in cellule di mammifero, 2 attività di PRMTs purificati, 3 Espressione e purificazione di substrato, e 4 completo trasferimento occidentale delle proteine ​​alla membrana di nitrocellulosa. <p cla...

Proteine ​​metilazione è stata descritta per la prima nel 1968 1. Fu solo la prima clonazione di PRMT1 nel 1996, che i ricercatori hanno cominciato ad apprezzare l&#39;importanza di questa modificazione post-traslazionale 2. È interessante notare che circa il 2% dei residui di arginina nelle proteine ​​di estratti nucleari sono metilato 3, che indica l&#39;abbondanza di questa modifica. L&#39;arginina è un aminoacido carico positivamente con una catena laterale di base ei azoto / s all&#39;interno delle catene laterali di arginina può essere post-traduzionali modificato mediante l&#39;aggiunta di un gruppo metile, un processo noto come arginina metilazione 4-6. Metilazione Arginina è catalizzata da una classe di enzimi viz., Transferasi proteine ​​metile arginina (PRMTs). Arginine possono sia essere monometilato o dimethylated la quale può essere sia simmetrica o asimmetrica a seconda del tipo di PRMTs catalizzano il processo 4-6. Le proteine ​​che visualizzano argininaricchi di motivi e-glicina sono potenziali bersagli per la catalisi PRMT-mediata. PRMT mediata da metilazione dei substrati è stato dimostrato di modulare l&#39;interazione proteina-proteina, l&#39;interazione proteina-acido nucleico, la funzione della proteina, l&#39;espressione genica, e / o di segnalazione cellulare, che sono tutti critici per la normale omeostasi cellulare 7-9. Al fine di comprendere il ruolo biologico delle proteine ​​arginina metilazione, precisi, efficienti e riproducibili test sono necessari per stabilire lo stato di metilazione dei substrati PRMT identificati. In vitro la metilazione, che valuta le capacità di PRMTs purificati per catalizzare metilazione dei loro substrati, è un saggio ben accettato per lo studio metilazione arginina 10-12. Il successo complessivo di questo test dipende in gran parte l&#39;attività dei PRMTs purificati. PRMTs possono essere espressi e purificati da batteri o cellule di mammifero 10,11. Questa metilazione test in vitro, come dNell&#39;odontoiatria nella sezione del protocollo, si basa su un metodo originariamente descritto da Tini e colleghi 10. In questo protocollo, vi mostriamo nel dettaglio i passaggi necessari per l&#39;espressione e la purificazione di PRMT1 in cellule di mammifero. La capacità del PRMT1 purificato per catalizzare metilazione su Ras-GTPasi attivando la proteina binding protein 1 (G3BP1), un noto substrato PRMT 8, è stato poi valutato in presenza di S-adenosil-L-[metil-3 H] metionina come donatore di metile. Usando questo test, possiamo definire in modo affidabile le capacità dei PRMTs di romanzo metilato o substrati noti, che è un passo fondamentale nello studio della proteina arginina metilazione.

<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

Proteina arginina metilazione è una delle più abbondanti modificazioni post-traduzionali nel nucleo. Proteine ​​arginina metilazione può essere identificato e / o determinato mediante approcci di proteomica, e / o immunoblotting con anticorpi specifici metil-arginina. Tuttavia, queste tecniche a volte possono essere fuorvianti e spesso fornire risultati falsi positivi. Soprattutto, queste tecniche non possono fornire prove dirette a sostegno della specificità di substrato PRMT. In vitro test di metilazione, d&#39;altra parte, sono saggi biochimici utili, che sono sensibili, e costantemente rivelano se le proteine ​​identificate sono davvero substrati PRMT. Un tipico test di metilazione in vitro comprende purificati, PRMTs attivi, substrato purificato e un radioisotopo marcato donatore di metile (S-adenosil-L-[metil-3 H] metionina). Qui si descrive un protocollo step-by-step per isolare PRMT1 cataliticamente attivo, un membro della famiglia PRMT ubiquitariamente espresso. Il meThyl attività transferasi del PRMT1 purificato sono stati successivamente testati su Ras-GTPasi attivando binding protein 1 (G3BP1) proteina, un substrato PRMT noto, in presenza di S-adenosil-L-[metil-3H] metionina come donatore di metile. Questo protocollo può essere impiegato non solo per stabilire lo stato di metilazione di nuovi substrati PRMT1 fisiologici, ma anche per comprendere il meccanismo di base della proteina arginina metilazione.

<html> Le capacità di HA-PRMT1 a metilato GST-G3BP1 sono stati determinati da un test in vitro metilazione in. PRMT1 HA-tag purificata da lisati cellulari Beas2B è stato impiegato in una reazione di metilazione contenente GST-G3BP1, e 1 μCi di S-adenosil-L-[metil-3 H] metionina, come donatore di metile. PRMT1 potrebbe efficacemente catalizzare la metilazione di GST-G3BP1 (Figura 1, pannello superiore). Reazioni di metilazione mediante pull-down eseguite su lisati di vuoto ...

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

Proteine ​​arginina metilazione, catalizzata da una classe di enzimi viz., Proteina arginina metil transferasi (PRMTs), è il processo di aggiunta enzimatica di gruppi metile (s) per arginines all&#39;interno di proteine. Il test in vitro metilazione è lo strumento più affidabile per valutare lo stato di metilazione di substrati PRMT noti o nuovi.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

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 Views.  University of Illinois at Chicago.  Proteine ​​arginina metilazione, catalizzata da una classe di enzimi viz., Proteina arginina metil transferasi (PRMTs), è il processo di aggiunta enzimatica di gruppi metile (s) per arginines all'interno di proteine. Il test in vitro metilazione è lo strumento più affidabile per valutare lo stato di metilazione di substrati PRMT noti o nuovi. 

Video: 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 di sensibilità visiva alla velocità e direzione del movimento in lucertole

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

Ricerca

Biologia

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.

Determinazione della metilazione del DNA di geni impressa Arabidopsis Dell&#39;endosperma

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.

Proteine ​​di membrana Overlay saggio: un protocollo di prova interazione tra proteine ​​solubili e insolubili 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.

Metilazione del DNA: Modifica bisolfito e analisi

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 liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

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.

Applicazione di un<em&gt; In vitro</em&gt; DNA Protection Assay per visualizzare stress Mediazione Proprietà del 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 ...

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.

Endoteliali Tubo cellulare saggio Formazione per la<em&gt; In Vitro</em&gt; Studio di Angiogenesi

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 ridotto Rappresentanza bisolfito sequenziamento per la valutazione della metilazione del DNA in Risoluzione coppia di basi

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.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

In vitro metilazione Assay per studiare proteine Arginina Metilazione