Name: monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting
Uploaded: 2011-10-17T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Bu çalışma Sağlık RO1-GM085130 Ulusal Enstitüsü ve Ulusal Bilim Vakfı MCB0929394 bağışları ile destek verdi. Dr. Marion Schmidt Biz onun misafirperverliği ve onun laboratuar film bize izin verdiği için teşekkür ediyorum.

1. Footprinting Reaktifler hazırlanması <ol><li> 100 mM sodyum cacodylate, 1 mM EDTA ve 1 M KCl içeren bir 10x reaksiyon tamponu hazırlayın. PH 7.4 &#39;e ayarlayın. Filtre 0.2 mcM asetat filtre cihazı (Nalgene) kullanarak tampon. Not: 10x tampon doğrudan pipetlemeyin RNA yok. </li><li> Tablo 1&#39;de gösterildiği gibi her bir reaksiyon için titrasyon reaksiyon karışımı hazırlayın. Titrasyon karışımı hacmi (1x istenilen konsantrasyonda tampon ve Mg (II)), 1x tampon RNA 10μl eklemeden önce, 90 ul...

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Hidroksil radikal footprinting nükleik asitlerin çözücü erişilebilir yüzey alanı değerlendirmek için değerli bir araçtır. Üçüncül yapısı 14 Kalitatif ve kantitatif oluşumu, iyon tipi ve konsantrasyonu, pH, sıcaklık, bağlayıcı proteinler veya katlama ko-faktörler gibi parametrelerin bir fonksiyonu olarak takip edilebilir . Yalındır ve ucuz bir protokol ve çıkan solvent erişilebilirlik ve tek nükleotid düzeyde katlanır bilgi zorlayıcı bir kombinasyon bu yöntem çok cazip hal...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA molekülleri, biyolojide önemli bir rol oynamaktadır. , RNA genetik bilginin iletilmesi için ek olarak, regülatör, bağlayıcı veya katalizör olarak belirli bir biyolojik rolünü yerine getiren benzersiz üçüncül yapıları içine katlayabilirsiniz. Üçüncül temas oluşumu hakkında bilgi RNA molekülleri işlevini anlamak için önemlidir. Hidroksil radikalleri (• OH), yüksek reaktivite ve küçük boyutları nedeniyle nükleik asitlerin yapısını benzersiz probları 1 footprinting prob olarak kullanıldığında, hidroksil radikalleri DNA 1 ve RNA&#39;nın 2 fosfodiester omurgası solvent erişilebilir yüzey haritası tek nükleotid çözünürlük gibi ince. Hidroksil radikal footprinting moleküller arası bir temas yüzeyi içerisinde nükleotidler, DNA-protein 1 ve RNA-protein komplekslerinin örneğin tanımlamak için kullanılır. Denge 3 ve 4 geçişler kinetik bir soluti bir fonksiyonu olarak hidroksil radikal footprinting yaparak tespit edilebilirdeğişken veya sırasıyla. Footprinting bir anahtar özelliği probu bu sınırlı maruz kalma (örneğin, &#39;tek-hit kinetiği), polimerin her bir nükleotid üniforması örnekleme sonuçları 5 Bu video makalede, RNA numune hazırlama ve Mg (II)-aracılı bir katlama izotermleri belirlenmesi göstermek için Tetrahymena ribozim P4-P6 etki alanı kullanın . H 2 O 2 (bu &#39;peroksidatif&#39; protokolünün diyoruz) ve doğal olarak çözünmüş O 2 kullanan bir değerli, ancak yaygın olarak bilinen, alternatif (biz bu &#39;gerektiren bilinen bir hidroksil radikal footprinting protokolü kullanımını tanımlamak oksidatif &#39;protokolü). Veri azaltma, dönüşüm ve analiz işlemleri genel bir bakış sunulmuştur.

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Bu protokol, Mg (II) bağlı hidroksil radikal footprinting iki yöntem RNA üçüncül yapısının oluşumu ölçmek için nasıl kullanılacağını açıklar.

RNA Yapısı &#39;peroksidatif&#39; ve &#39;Oksidatif&#39; Hidroksil Radikal footprinting İzleme Denge Değişiklikler

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

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