Diese Studie wurde von einem Merit Award des US Department of Veterans Affairs unterstützt, und ein NIH Zuschusses R01CA138528 Rw.

1. Herstellung der Expressionskonstrukte <ol><li> Vor dem Starten des Protokolls Klon PRMTs in Säugetier-Expressionsvektoren mit einer N-terminalen Epitop-Tag (Myc oder HA), und das Substrat in-frame mit Glutathion-S-Transferase (GST) für High-Level-Proteinexpression und Aufreinigung von Bakterienzellen Lysaten unter Verwendung von molekularbiologischen Standardtechniken. </li></ol> 2. Reinigung von PRMTs <ol><li> Verwenden Sie 100 mm Kulturschalen für die menschliche bronchialen Epithelzellen (Beas2B) zu halten. Durchgang die Zellen alle 3 Tage und lassen Sie sie nicht zu Zusammenfluss wachsen. </li><li> Am Tag vor der Transfektion Saatgut 7,5 x 10 5 Zellen in 10 ml Kulturmedium in einer 100 mm Kulturschale. Wirbel um gleichmäßig zu verteilen, die Zellen, und bei 37 ° C in einer 5% CO 2 befeuchteten Inkubator. </li><li> Am nächsten Tag, aspirieren die Medien und mit 5 ml Kulturmedium ohne Serum und Antibiotika. Wirbeln und saugen das Medium. Wiederholen Sie diesen Schritt einmehr Zeit, und schließlich mit 5 ml Serum / Antibiotika-freiem Medium, und fahren Sie Transfektion. </li><li> Verdünnen 6 ug Plasmid-DNA in 750 ul serumfreiem Medium in einem sterilen 1,5 ml-Plastikzentrifugenröhrchen. </li><li> In einer weiteren 1,5 ml-Plastikzentrifugenröhrchen verdünnt 30 ul Transfektionsreagenz in 750 ul serumfreiem Medium. </li><li> Übertragen Sie die verdünnte Lösung Transfektionsreagenz (Schritt 2.5), um das Röhrchen mit DNA verdünnt (Schritt 2.4). Vorsichtig mischen und Abpipettieren mehrmals. </li><li> Inkubieren der Transfektion Mischung bei Raumtemperatur für 15 min. </li><li> Nach 15 min, Pipette die Transfektion Mischung sanft auf die Zellen, Wirbel gleichmäßig zu mischen, und bei 37 ° C in einer 5% CO 2 befeuchteten Inkubator. </li><li> Nach 3 Stunden absaugen Medium mit Transfektion Mischungen aus den Zellen. Fügen Sie frisches Zellkulturmedium mit Serum und Antibiotika ergänzt. </li><li> Bei 37 ° C in einem befeuchteten 5% CO 2Inkubator für 48 Stunden. </li><li> Nach 48 Stunden lysieren die Zellen in 1 ml Lyse-Puffer (1x phosphatgepufferter Kochsalzlösung, 1% Nonidet P-40, 0,5% Natriumdesoxycholat, 0,1% Natriumdodecylsulfat und 1 ug / ml phenlymethyl Sulfonylfluoridform). Sammle die Lysate in ein Kunststoffzentrifugenröhrchen mit einem Zellheber und Inkubation der Lysate auf Eis für 30 min. </li><li> Zentrifugieren Sie die Lysate bei 15.000 g für 10 min auf einer Tischzentrifuge, um die Lysate von Zelltrümmer zu entfernen. [* Es wird dringend empfohlen, die Expression von HA-PRMT1 über Immunoblotting eines Aliquots der Lysate mit Anti-HA-Antikörper, bevor Sie fortfahren, um die Reinigung der Enzyme für Methylierungstest bestätigen.] </li><li> Zur Reinigung von PRMTs inkubieren 3 mg von Zelllysaten mit 30 ul 50:50 Suspension von Anti-HA-Affinitätsmatrix in phosphatgepufferter Salzlösung (PBS), und drehbar über Nacht bei 4 ° C in einem Rohrdrehgelenk. </li><li> Nächsten Tag drehen die Rohre bei 14.000 xg auf einer Tischzentrifuge für 2 min zu sammelndie Perlen. Überstand verwerfen und Waschen der Kügelchen 3x mit 1 ml RIPA-Puffer [20 mM Tris (pH 8,0), 150 mM NaCl, 5 mM EDTA und 1% Triton-X-100] in jedem Waschen durch Zentrifugation bei 14.000 g für 2 Minute Nach dem dritten Waschen, Waschen der Kügelchen einmal mit 100 ul Methylierungspuffer (50 mM Tris pH 8,5, 20 mM KCl, 10 mM MgCl 2, 1 mM β-Mercaptoethanol, 100 mM Saccharose). Nach der Zentrifugation den Überstand entfernen. Die Perlen, die die immobilisiert PRMTs sind bereit, bei der Methylierung Assay verwendet werden. [* Es wird dringend empfohlen, die immobilisiert PRMTs sofort in der Methylierungstest zu verwenden.] </li></ol> 3. Reinigung der Substrat <ol><li> Inokulieren eine einzelne Kolonie von GST-G3BP1 transformierten BL21 in 5 ml Luria-Bertani (LB)-Medium, enthaltend 100 ug / ml Ampicillin. Wachsen die Kulturen über Nacht bei 37 ° C mit Schütteln bei 250 UpM. </li><li> Am nächsten Tag überweisen Sie den Nacht-Kultur auf 50 ml frisches LB-Medium und Fortsetzunginue zu einer OD 600 von 0,5-0,7 wachsen (es dauert ca. 2 h). </li><li> Wenn die OD 600 0,5-0,7 erreicht, fügen Isopropyl-β-D-thiogalactosid (IPTG) in einer Endkonzentration von 1 mM und weiterhin Kultur für weitere 4 Stunden bei 37 ° C mit Schütteln bei 250 UpM. </li><li> Ernte der Zellen durch Zentrifugation bei 1.500 g für 10 min und verarbeiten das Pellet für GST Reinigung oder bei -80 ° C. </li><li> Resuspendieren der Bakterienpellet in 5 ml Lyse-Puffer (1 × PBS mit Proteaseinhibitoren, 100 ug / ml Lysozym und 0,1% Triton-X-100) und Inkubation der Lysate bei 37 ° C für 30 min. Zu diesem Zeitpunkt werden die Lysate trübe und viskos aufgrund der Freisetzung der bakteriellen genomischen DNA auftreten. </li><li> Scherung der genomischen DNA durch Ultraschallbehandlung (10 Impulse jeder mit einer Amplitude von 30% 1 sec). </li><li> Deaktivieren Sie die Lysate durch Zentrifugation bei 15.000 g für 10 min und übertragen Sie die klare Überstand in ein 15-ml-Tube. Wenn der Überstand ist nicht klar, die centrifu wiederholengation Prozess noch einmal. </li><li> Inzwischen, waschen Sie die GST-Sepharose-Kügelchen zweimal mit eiskaltem PBS und eine 50:50 Aussetzung der GST-Sepharose in PBS. Jeweils 200 ul des GST-Sepharose-Suspension auf die frei bakteriellen Zelllysat und rotieren bei 4 ° C für 1 Stunde. </li><li> Nach 1 Stunde Zentrifugation der Röhrchen bei 500 g für 3 min, um die Perlen zu sammeln. Waschen der Kügelchen 3x mit eiskaltem PBS. </li><li> Nach dem letzten Waschen, Resuspendieren der Kügelchen in 100 ul Elutionspuffer (50 mM Tris pH 8,0, 150 mM NaCl, 30 mM Glutathion) und drehen sich bei 4 ° C für 10 min. </li><li> Zentrifugieren Sie die Röhrchen bei 15.000 g für 2 min und den Überstand der gereinigten Protein enthält, in einem anderen Kunststoffzentrifugenröhrchen. Durchführung der SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE)-Analyse von 5 ul des gereinigten Proteins mit bekannten Mengen an Rinderserumalbumin (BSA), um die Konzentration des gereinigten Proteins zu bestimmen. </li></ol> 4. Methylierung Assay HINWEIS: Führen Sie folgende Schritte in einem Radioisotop für Arbeit und in Übereinstimmung mit den Protokollen des Instituts auf die Durchführung von Experimenten mit radioaktiven Isotopen bezeichnet separaten Bereich. <ol><li> Add 2 ug von gereinigtem GST-Substrat und einer Master-Mix, enthaltend 1x Methylierungspuffer (50 mM Tris pH 8,5, 20 mM KCl, 10 mM MgCl 2, 1 mM β-Mercaptoethanol, 100 mM Saccharose), 1 uCi S-Adenosyl L-[Methyl-3 H]-Methionin und Wasser auf ein Endvolumen von 10 ul zu den immobilisierten PRMTs (Schritt 2.13), und Inkubieren der Reaktion bei 30 ° C für 1 Stunde. </li><li> Nach 1 Stunde, stoppen Sie die Reaktion durch Zugabe von 10 ul 2x SDS-Probenpuffer. Nach der Denaturierung der Proben bei 95 ° C für 10 min, Trennen der Proben auf einem SDS-PAGE-Gel. </li><li> Später übertragen die aufgetrennten Proteine ​​aus dem SDS-Gel auf eine Nitrocellulosemembran unter Verwendung eines halbtrockenen elektrophoretischen Transfer. </li><li> Nach der Übertragung umfassen die Membran mit einemutofluor (eine autoradiographische Bildverstärker) und schaukeln für 2 Stunden bei Raumtemperatur. Nach 2 Stunden, abgießen autofluor und behalten für die spätere Verwendung. [* Das Röhrchen als radioaktives Material] </li><li> Luft trocknen schnell die Membran auf ein Filterpapier, versiegeln in einem Kunststoffbeutel und setzen die Membran auf einem Röntgenfilm bei -20 ° C. Ausreichende Belichtungszeit liegt zwischen 5-30 Tagen. </li></ol> 5. Bestätigung des PRMT Expression und gleichmäßige Beladung des Substrats <ol><li> Nach Erreichen einer ausreichenden Engagements, übertragen Sie die Membran auf eine Kunststofffenster mit Blocking-Puffer [Tris-gepufferte Kochsalzlösung mit Tween-20 (TBST + 3% fettfreier Milch)], und schaukeln für 1 Stunde. </li><li> Entsorgen Sie die Blockierungspuffer als radioaktiver Abfall. Inkubieren der Membran mit anti-HA-Antikörper (1: 1000 Verdünnung) in TBST über Nacht bei 4 ° C, unter leichtem Schütteln. </li><li> Waschen Sie die Membran mit TBST 3x und Inkubation der Membran mit HRP-markierte Anti-Maus-Sekundärantikörper(1: 5000 Verdünnung) in TBST bei Raumtemperatur für 1 Stunde unter leichtem Schütteln. </li><li> Nach 1 Stunde, waschen Sie die Membran mit TBST 3x, und führen Chemilumineszenz Detektion von Proteinen mit einem HRP-Substrat. </li><li> Nach Erkennen des HA-markierten PRMTs, Inkubation der Membran mit Ponseau-S-Färbelösung für 5 min bei Raumtemperatur. Waschen schnell die Membran 3x mit Wasser, um die GST-markierten Substraten zu erkennen. Scannen Sie das Bild. </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>

Die Autoren haben nichts zu offenbaren.

Das hierin beschriebene Protokoll wird routinemäßig verwendet, um den Methylierungsstatus der identifizierten PRMT Substraten herzustellen. Diese robuste und konsistente Assay wird auch definitive Beweise für die Spezifität der PRMTs für ihre Substrate. Die Schlüsselkomponenten für den Erfolg dieses Tests sind: 1. Die Expression von PRMTs in Säugerzellen, 2. Aktivität von gereinigtem PRMTs, 3. Expression und Reinigung des Substrats, und 4. Vollständige westlichen Transfer der Proteine ​​auf die Nitrocellul...

Protein-Methylierung wurde erstmals im Jahre 1968 beschrieben 1. Es war nicht bis zum ersten Klonen von PRMT1 im Jahr 1996, dass die Forscher begannen, die Bedeutung dieser post-translationale Modifikation 2 zu schätzen wissen. Interessanterweise etwa 2% der Argininreste in den Proteinen des Zellkernextrakten methyliert 3, was die Häufigkeit dieser Modifikation. Arginin ist eine positiv geladene Aminosäure mit basischer Seitenkette und der Stickstoff / s in den Seitenketten von Arginin kann posttranslational durch die Zugabe einer Methylgruppe, einem Verfahren, wie Arginin Methylierung 4-6 bekannt sind, modifiziert. Arginin-Methylierung wird durch eine Klasse von Enzymen katalysiert nämlich., Protein-Arginin-Methyltransferasen (PRMTs). Arginine entweder monomethylierte oder dimethyliert, und letztere kann entweder symmetrisch oder asymmetrisch sein, abhängig von der Art des PRMTs Katalyse der Prozess 4-6. Proteine, die Arginin anzeigenE-Glycin-reiche Motive sind mögliche Ziele für PRMT-vermittelten Katalyse. PRMT-vermittelte Methylierung von Substraten wurde gezeigt, dass Protein-Protein-Wechselwirkung, Protein-Nukleinsäure-Wechselwirkung, Proteinfunktion, Genexpression und / oder zelluläre Signal, die alle für die normale zelluläre Homöostase 7-9 modulieren. Um die biologische Rolle von Protein-Arginin-Methylierung zu verstehen, werden genaue, effiziente und reproduzierbare Tests erforderlich, um den Methylierungsstatus der identifizierten PRMT Substraten herzustellen. In-vitro-Methylierungsassay, der die Fähigkeiten des gereinigten PRMTs ausgewertet Methylierung ihrer Substrate katalysieren, ist eine gut akzeptierte Assays zur Untersuchung Arginin Methylierung 10-12. Der Gesamterfolg dieses Tests hängt weitgehend von der Aktivität der gereinigten PRMTs. PRMTs exprimiert und aus Bakterien oder Säugetierzellen 10,11, gereinigt werden. Diese in-vitro-Methylierungsassay, wie din der Protokollabschnitt etailed, basiert auf einer Methode, die ursprünglich von Tini und Kollegen 10 beschrieben basiert. In diesem Protokoll zeigen wir im Detail die Schritte in der Expression und Reinigung von PRMT1 in Säugerzellen. Die Fähigkeit des gereinigten PRMT1 Methylierung Ras-GTPase-aktivierendes Protein katalysieren binding protein 1 (G3BP1), einem bekannten PRMT Substrat 8 wurde später in der Gegenwart von S-Adenosyl-L-[Methyl-3 H]-Methionin als ermittelten Methyldonor. Unter Verwendung dieses Tests können wir zuverlässig die Fähigkeiten der PRMTs definieren Methylat neuen oder bekannten Substrate, die eine primäre Schritt bei der Untersuchung von Protein-Arginin-Methylierung ist.

<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

Protein-Arginin-Methylierung ist eine der am häufigsten vorkommenden posttranslationalen Modifikationen in den Zellkern. Protein-Arginin-Methylierung können identifiziert und / oder durch proteomische Ansätze bestimmt und / oder Immunoblotting mit Methyl-Arginin-spezifischen Antikörpern. Diese Techniken können manchmal irreführend sein und oftmals falsch positive Ergebnisse. Am wichtigsten ist, können diese Techniken nicht einen direkten Beweis zur Unterstützung der PRMT Substratspezifität. In vitro Methylierung Assays auf der anderen Seite, sind nützlich biochemische Assays, die empfindlich sind und durchweg zeigen, ob die identifizierten Proteine ​​sind in der Tat PRMT Substraten. Eine typische in vitro-Methylierungsassay enthält gereinigtes, aktives PRMTs gereinigte Substrat und einem Radioisotop markiert Methyldonors (S-Adenosyl-L-[Methyl-3 H]-Methionin). Hier beschreiben wir eine Schritt-für-Schritt-Protokoll zu katalytisch aktiven PRMT1, ein ubiquitär exprimiert PRMT Familienmitglied zu isolieren. Die michthyl-Transferase-Aktivitäten der gereinigten PRMT1 wurden später auf Ras-GTPase-aktivierendes Protein-Bindungsprotein 1 (G3BP1), einem bekannten PRMT Substrat getestet, in Gegenwart von S-Adenosyl-L-[Methyl-3 H] Methionin als Methyl-Donor. Dieses Protokoll kann nicht nur zur Bestimmung des Methylierungsstatus von neuen physiologischen PRMT1 Substrate, sondern auch für das Verständnis des grundlegenden Mechanismus der Protein-Arginin-Methylierung eingesetzt werden.

<html> Die Fähigkeit von HA-PRMT1 GST-G3BP1 methylieren wurden durch einen in-vitro-Methylierungsassay bestimmt. HA-getaggte PRMT1 von Beas2B Zelllysate gereinigt wurde, in einem Methylierungsreaktion enthaltende GST-G3BP1 verwendet und 1 uCi von S-Adenosyl-L-[Methyl-3 H]-Methionin als Methyl-Donor. PRMT1 könnte effizient katalysieren die Methylierung von GST-G3BP1 (Abbildung 1, oben). Methylierungsreaktionen mit Pulldown-Menüs auf Lysaten von leeren Vektor transfizierten Zellen Be...

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

Protein-Arginin-Methylierung durch eine Klasse von Enzymen katalysiert viz., Protein-Arginin-Methyltransferasen (PRMTs), ist das Verfahren der enzymatischen Addition von Methyl-Gruppe (n) Arginine in Proteinen. Die in-vitro-Methylierungstest ist die zuverlässigste Werkzeug für die Beurteilung der Methylierungsstatus von bekannten oder neuen PRMT Substraten.

In-vitro-Test zur Methylierung Protein-Arginin-Methylierung Studieren

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

in-vitro-methylation-assay-to-study-protein-arginine-methylation

Research

JoVE Journal

Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

15.1K Aufrufe.  University of Illinois at Chicago.  Protein-Arginin-Methylierung durch eine Klasse von Enzymen katalysiert viz., Protein-Arginin-Methyltransferasen (PRMTs), ist das Verfahren der enzymatischen Addition von Methyl-Gruppe (n) Arginine in Proteinen. Die in-vitro-Methylierungstest ist die zuverlässigste Werkzeug für die Beurteilung der Methylierungsstatus von bekannten oder neuen PRMT Substraten. 

Serie: 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.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

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

Forschung

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.

Bestimmung der DNA-Methylierung von geprägten Genen 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 ...

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.

Proteinmembran Overlay Assay: Ein Protokoll, die Interaktion zwischen löslichen und unlöslichen Proteinen testen 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.

DNA-Methylierung: Bisulfit Modifikation und 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.

Eine flüssige Phase Affinity Capture Assay Verwendung von magnetischen Kügelchen, um Protein-Protein Interaktion Studie: Das Poliovirus-Nanobody Beispiel

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.

Anwendung eines<em&gt; In-vitro-</em&gt; DNA-Schutz-Assay, um Stress Mediation Eigenschaften des DPS-Protein Visualisieren

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.

Endothelzellen Schlauchbildung Assay für die<em&gt; In-vitro-</em&gt; Studium der Angiogenese

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.

Verbesserte Reduzierte Darstellung Bisulfit-Sequenzierung für die Bewertung der DNA-Methylierung bei Basenpaar Auflösung

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.