Name: horizontal gel electrophoresis for enhanced detection of protein-rna complexes
Uploaded: 2017-07-28T14:00:00
Description: Watch this Scientific Journal Video about Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes at JoVE.com

Rakamlar hazırladığımız için Laura Vanderploeg&#39;e teşekkür ediyoruz. E-Tablolar laboratuarı çalışması, NSF hibe 1050395 ve NIH hibe (R21HD076828) tarafından desteklenmektedir. Ryder laboratuarı NIH hibe R01GM117237 ve R01GM117008 tarafından desteklenmektedir. Megan Dowdle, National Institutes of Health&#39;in Ulusal Tıbbı Bilimler Ulusal Enstitüsü (T32GM008349) aracılığıyla Wisconsin-Madison Enstitüsü ve Biyoteknoloji Eğitim Programı aracılığıyla SciMed GRS İleri Fırsat Fellowitesi tarafından desteklenmektedir.

1. Yatay Native Poliakrilamid Jelin Hazırlanması 4 . <ol><li> Gerekli malzemelerin hazırlanması. <ol><li> Yatay jel aparatı için 27 cm x 21 cm&#39;lik bir tepsi ve iki adet 24 kuyucuklu tarak için bir jel kutusu (37 cm x 24 cm) kullanın. Bu kurulum, aynı anda analiz edilebilecek toplam 48 örnek sağlar. </li><li> Aşağıdaki reaktifleri hazırlayın:% 40 Acrylamide-Bis 19: 1, 5x TBE (Tris-Borat-EDTA pH 8.0), TEMED ve% 10 APS (amonyum persülfat). </li></ol></li><li> ...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+RNA+binding+and+translational+repression+by+the+Bicaudal-C+regulatory+protein.">Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.</a> J Biol Chem. 289 (11), 7497-7504 (2014).</li><li>Gamberi, C., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bic-C+family+of+developmental+translational+regulators.">The Bic-C family of developmental translational regulators.</a> Comp Funct Genomics. , 141386 (2012).</li><li>Saffman, E. 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Yerli poliakrilamid jelleri, protein-RNA etkileşimlerini araştırmak için paha biçilemez bir araçtır ve geleneksel olarak bu jeller dikey olarak 2 , 3 elektroforez edilir. Doğal poliakrilamid jelleri oluşturan ve yatay olarak elektroforez edilen 1 , 4 , 6 , 7 , 15&#39;in yerini alan bir protok...

Elektroforetik mobilite kayma testleri (EMSA&#39;lar), spesifik protein-nükleik asit etkileşimleri 1 , 2 , 3&#39;ü analiz etmek için paha biçilemez bir biyokimyasal araç olduğu kanıtlanmıştır. Bu tahliller, proteinlerin RNA veya DNA 3&#39;e bağlanma afinitesi hakkında önemli bilgi sağlayabilir ve nükleik asit-protein komplekslerinin 1 bileşen stokiyometrisi 1 ve substrat rekabet deneyleri 1 yoluyla RNA bağlama proteinlerinin bağlanma spesifitesi hakkında önemli yeni bilgiler sağlar 1 . Bu deneyler için geleneksel deney düzeneği, saflaştırılmış proteinin radyo-etiketli bir RNA substratı ile karıştırılmasından oluşur. Ortaya çıkan kompleksler daha sonra iki cam levha arasına dökülen denatüre edici olmayan (doğal) poliakrilamid jeller ile, ardından dikey bir cihazda 3 numune elektroforeziyle analiz edilir. Bu yaklaşım, proteinlerin nükleik asitlere bağlanmasının temelini oluşturan biyokimyasal mekanizmalarda önemli bilgiler sağlamak için kapsamlı bir şekilde kullanılmış olmakla birlikte, çeşitli sınırlamaları da vardır. Örneğin, bu temel strateji nispeten düşük işlem hacmine sahiptir ve birçok bağlama reaksiyonunu paralel olarak analiz etmeyi gerektiren uygulamalar için kolayca uyarlanamaz. Buna ek olarak, geleneksel dikey aparat ile elektroforez 3 , 4 sırasında kompleksleri birden çok kez potansiyel olarak izlemek zor. Burada, düz yataklı bir aparatta, yatay elektroforezde ve floresanla etiketlenmiş RNA substratları 4 , 5 , 6 , 7&#39;de yerli poliakrilamid jelleri kullanan EMSA tahlilinin uyarlamasını sunuyoruz. Bu nispeten basit değişikliklerin temel samana dahil edilmesiTegy bazı güçlü avantajlar sağlar. Özellikle yatay masaüstü biçimi, düzinelerce numuneyi eşzamanlı olarak analiz etmeye yardımcı olur 4 . Ayrıca, Bicaudal-C proteini ile RNA substratı arasında oluşan RNA-protein kompleksleri için, yatay bir jelde elektroforez farklı RNA-protein komplekslerini çözmek ve bağlanmamış RNA substrattan ayırmak için artan bir yeteneğe sahiptir.

<table border="0" cellpadding="0" cellspacing="0" width="1432">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Horizontal Gel Box</td>
			<td style="width:112px;">OWL</td>
			<td style="width:116px;">N/A</td>
			<td style="width:831px;">Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel boxes. Catalog Number: A2-BP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well large large horizontal gel electrophoresis combs</td>
			<td>OWL</td>
			<td>N/A</td>
			<td>Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel box combs. Catalog Number: A2-24C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerpac 300</td>
			<td>Bio-Rad</td>
			<td>1655050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Protean II Electrophoresis Cell</td>
			<td>Bio-Rad</td>
			<td>165-2940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InstaPAGE-19 40% 19:1 Acrylamide/Bis</td>
			<td>IBI</td>
			<td>IB70015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>IBI</td>
			<td>IB70120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APS</td>
			<td>IBI</td>
			<td>IB70080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNAs</td>
			<td>Ambion</td>
			<td>AM7119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescein labeled RNA</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Order can be made custom to length and desired sequence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA tetrasodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>E5391-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base Ultrapure</td>
			<td>US Biological</td>
			<td>T8600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>BP168-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEPC</td>
			<td>Sigma-Aldrich</td>
			<td>1609-47-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>3483 12 3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

horizontal gel electrophoresis for enhanced detection of protein-rna complexes

Yerli poliakrilamid jel elektroforezi, RNA-protein etkileşimlerinin biyokimyasal analizi için kapsamlı bir şekilde kullanılan moleküler biyolojinin temel bir araçtır. Bu etkileşimler geleneksel olarak iki cam levha arasında üretilen poliakrilamid jelleri ve numuneler dikey olarak elektroforez ile analiz edilmiştir. Bununla birlikte, tepsilerde dökülen ve yatay olarak elektroforez edilen poliakrilamid jeller çeşitli avantajlar sunmaktadır. Örneğin, flüoresans RNA substratları ve spesifik proteinler arasındaki kompleksleri analiz etmek için kullanılan yatay jeller, elektroforez ilerledikçe birçok kez görüntülene edilebilir. Bu, deney sırasında RNA-protein komplekslerini çeşitli noktalarda izlemek için benzersiz bir fırsat sağlar. Buna ek olarak, yatay jel elektroforezi birçok numuneyi paralel olarak analiz etmeyi mümkün kılar. Bu, farklı bileşenleri ve koşulları karşılaştırmak için zamanlama deneylerini büyük ölçüde kolaylaştırabilir ve aynı anda birden fazla reaksiyonu analiz edebilir. İşte biz prRNA-Protein etkileşimlerini analiz etmek için yatay doğal jel elektroforezi üretmek ve kullanmak için ayrıntılı bir protokolü inceleyin.

Yatay doğal jel elektroforezinin gücü ve çok yönlülüğünü göstermek için Xenopus Bicaudal-C (Bicc1) proteininin bir Bicc1 bağlanma yeri içeren floresanla işaretlenmiş bir RNA&#39;ya bağlandığını analiz ettik. Bicc1 proteinleri, hayvanlarda gelişme 9 , 10 , 11 , 12&#39;nin maternal evreleri boyunca hücre akıbeti kararlarını kont...

Watch this Scientific Journal Video about Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes at JoVE.com

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Doğal poliakrilamid jel elektroforezi, RNA-protein etkileşimlerini analiz etmek için temel bir araçtır. Geleneksel olarak çoğu deney dikey jel kullanmıştır. Bununla birlikte, yatay jeller elektroforez sırasında kompleksleri izleme fırsatı gibi birçok avantaj sağlar. Yatay doğal jel elektroforezinin üretilmesi ve kullanılması için ayrıntılı bir protokol sağlıyoruz.

Protein-RNA Komplekslerinin Geliştirilmiş Algılaması için Yatay Jel Elektroforezi

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

The overall of this protocol is to analyze RNA protein interactions using horizontal native gel electrophoresis. This method can help answer key questions in RNA biochemistry, such as defining the characteristics of specific RNA protein interactions. The main advantage of this technique is that it allows complex formation to be monitored multiple times during electrophoresis.For the horizontal gel apparatus, use a gel box with a 27 centimeter by 21 centimeter casting tray and a capacity for two 24 well combs. This set up provides a total of 48 samples that can be analyzed simultaneously. Prepare two liters of 1x TBE running buffer by diluting 400 milliliters of 5x TBE with 1, 600 milliliters of water and mixing.Place the TBE buffer in the cold room to cool. Prepare the following reagents following standard procedures, 40%acrylamide best 19:1, 5x TBE, temed and 10%ammonium per sulfate. To make a 10%native acrylamide gel, add the following to a 500 milliliter flask:80 milliliters of 5x TBE, 100 milliliters of 40%acrylamide and water to a final volume of 400 milliliters.Add four milliliters of 10%ammonium per sulfate and 400 microliters of temed and mix well. Pour the mix into the horizontal casting tray and allow the gel to polymerize in the fume hood for 30 minutes. The polymerized gel will be approximately two centimeters thick and a thin layer of liquid will remain on top of the gel.Place the gel into the gel box and take it into the cold room. Next, pour the liquid off and rinse with running buffer. Remove the combs carefully, add two liters of cold running buffer to the gel box and then use a syringe to rinse the wells with running buffer.Pre-run the gel at 120 volts for one hour at four degrees celsius in a cold room. While the gel is being prerun, prepare the binding reactions. Add the following binding reaction components to each RNA free microcentrifuge tube in this order:five microliters of 10 millimolar BSA, five microliters of 20 millimolar DTT, five microliters of 10 millimolar yeast tRNAs, 10 microliters of 5x binding buffer, bicaudal-c protein to a final concentration of 250 nanomolar in 50 microliters, DEPC-treated water to a final volume of 45 microliters and five microliters of fluorescently labeled RNA substrate.Close the tubes and flick gently to mix, centrifuge the tubes briefly and incubate in the dark for 30 minutes at room temperature. When the binding reactions are complete, add 15 microliters of 50%glycerol to each reaction and mix gently. Carefully load 30 microliters of each reaction into the wells of the gel, the amount of sample that can be loaded into a single well varies depending on the thickness of the gel and the size of the wells.The power supply to the gel apparatus, switch on the power, adjust the voltage to 120 volts and run the gel in four degree celsius. To prevent damaging or bleaching of the fluorescently labeled RNA, conduct electrophoresis in the dark. Electrophoresis times will vary depending upon the specifics of the RNA protein complex being analyzed.A major advantage of the horizontal electrophorated mobility shift assay analysis, is that electrophoresis can be stopped in the gel image prior to completion of the run. Perform analysis with any instrument designed for detecting an imaging fluorescent substrates. Adjust the imager settings, for fluorescently labeled ligands, use the following settings.Fluorescein wavelength of 473 nanometers, photomultiplier voltage of 750 volts and pixel size of 100 micrometers. Scan the gel to capture the image. After imaging, the gel can be placed back into the apparatus and electrophoresis can be continued for longer times.The binding of the xenopus bicaudal-C or Bicc1 protein to a fluorescently labeled Cripto1 mRNA containing a Bicc1 binding site, was analyzed by both vertical and horizontal native gel electrophoresis, as a negative control, Bicc1 was mixed with a fluorescently labeled RNA derived from CyclinB1 mRNA. It was readily apparent using either method, that Bicc1 binds to the Cripto1 RNA and forms a specific complex. However, the Bicc1, Cripto1 complexes were significantly more distinct when analyzed with the horizontal gel, facilitating their detection and allowing the discrimination of protein RNA complexes from unbound RNA.After imaging, the gel was returned to the gel apparatus, electrophoresis for an additional three and a half hours, and reimaged. As shown here, the complexes had migrated further and were still readily detectable, indicating their stability. Once mastered, this technique can be done in four to five hours, if it is performed properly.After watching this video, you should have a good understanding of how to analyze RNA protein interactions using horizontal native gel electrophoresis.

Research

JoVE Journal

Biochemistry

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

Biyolojik Numune metabolik profil için Sheathless Kapiler Elektroforez-Kütle Spektrometre

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Biyokimya

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

Denatüre edici olmayan poliakrilamid jel elektroforez kullanılarak insan mxa Çok alt birim protein kompleksleri karakterizasyonu

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Ökaryotik Hücrelerde Protein Kompleksleri Tandem Affinity Arıtma

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Analiz için Protein-tRNA Agaroz Jel Retardasyon Testleri<em&gt; K</em<sup&gt; 6</sup&gt; Threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Proximity Ligation Testi ile Süspansiyon Hücre Kültürlerinde DNA Hasarına Bağlı Protein Komplekslerinin Saptanması ve Görselleştirilmesi

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Mitokondriyal Elektron Nakil Zincirinin Süper Komplekslerinin Yerli Elektroforez, In-Jel Testleri ve Elektroelüsyon ile İncelenmesi

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

DNA polimeraz etkinliği tahlil Akrilamid Jel Elektroforez tarafından görüntülenmiştir yakın kızılötesi floresan etiketli DNA'yı kullanarak

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

İki boyutlu yarı denaturing deterjan özel Jel Elektroforez amiloid veya amiloid benzeri liflerin boyutu heterojen onaylamak için kullanımı

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

AtHIRD11 içsel bozukluğu ve bağlama kapiller Jel Elektroforez ve benzeşme kapiler Elektroforez Metal iyonları doğru Analizi

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Thylakoid membran Protein kompleksleri tarafından mavi yerli jel elektroforez Analizi

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...