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

Vi takker Laura Vanderploeg for at forberede tal. Arbejde i Sheets lab understøttes af NSF grant 1050395 og NIH grant (R21HD076828). Arbejde i Ryder-labet understøttes af NIH-tilskud R01GM117237 og R01GM117008. Megan Dowdle støttes af et SciMed GRS Advanced Opportunity Fellowship gennem University of Wisconsin-Madison Graduate School og Bioteknologi Training Program gennem National Institute of General Medical Sciences af National Institutes of Health (T32GM008349).

1. Fremstilling af den horisontale indfødte polyacrylamidgel 4 . <ol><li> Forberedelse af de nødvendige materialer. <ol><li> Til det vandrette gelapparat skal du bruge en gelkasse (37 cm x 24 cm) med en 27 cm x 21 cm bakke og en kapacitet til to 24 brønde kamme. Denne opsætning giver i alt 48 prøver, der kan analyseres samtidigt. </li><li> Forbered følgende reagenser: 40% Acrylamide-Bis 19: 1, 5x TBE (Tris-Borat-EDTA pH 8,0), TEMED og 10% APS (ammoniumpersulfat). </li></o...

<ol>
	<li>Ryder, S. P., Recht, M. I., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift.">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li><li>Dahlberg, A. E., Dingman, C. W., Peacock, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+characterization+of+bacterial+polyribosomes+in+agarose-acrylamide+composite+gels.">Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels.</a> J Mol Biol. 41 (1), 139-147 (1969).</li><li>Hellman, L. M., Fried, M. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Characterization+of+Binding+Oxytricha+nova+Single+Strand+Telomere+DNA+with+the+Alpha+Protein+N-terminal+Domain.">Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain.</a> J Mol Biol. 359 (5), 1217-1234 (2006).</li><li>Zhang, Y., Park, S., Blaser, S., Sheets, M. 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. E., 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=Premature+translation+of+oskar+in+oocytes+lacking+the+RNA-binding+protein+bicaudal-C.">Premature translation of oskar in oocytes lacking the RNA-binding protein bicaudal-C.</a> Mol Cell Biol. 18 (8), 4855-4862 (1998).</li><li>Chicoine, J., 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=Bicaudal-C+recruits+CCR4-NOT+deadenylase+to+target+mRNAs+and+regulates+oogenesis,+cytoskeletal+organization,+and+its+own+expression.">Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression.</a> Dev Cell. 13 (5), 691-704 (2007).</li><li>Zhang, Y., 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=Bicaudal-C+spatially+controls+translation+of+vertebrate+maternal+mRNAs.">Bicaudal-C spatially controls translation of vertebrate maternal mRNAs.</a> RNA. 19 (11), 1575-1582 (2013).</li><li>Maisonneuve, C., 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=Bicaudal+C,+a+novel+regulator+of+Dvl+signaling+abutting+RNA-processing+bodies,+controls+cilia+orientation+and+leftward+flow.">Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow.</a> Development. 136 (17), 3019-3030 (2009).</li><li>Tran, U., 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=The+RNA-binding+protein+bicaudal+C+regulates+polycystin+2+in+the+kidney+by+antagonizing+miR-17+activity.">The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.</a> Development. 137 (7), 1107-1116 (2010).</li><li>Pagano, J. 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Indfødte polyacrylamidgeler er et uvurderligt redskab til undersøgelse af protein-RNA-interaktioner, og traditionelt er disse geler elektroforeseret vertikalt 2 , 3 . Vi har anvendt en modifikation af protokollen, som erstatter native polyacrylamidgeler, der er oprettet og elektroforeseret vandret 1 , 4 , 6 , 7 , <sup clas...

Elektroforetiske mobilitetsforskydningsassays (EMSA&#39;er) har vist sig at være et uvurderligt biokemisk værktøj til analyse af specifikke protein-nucleinsyre-interaktioner 1 , 2 , 3 . Disse analyser kan tilvejebringe vigtig information vedrørende proteinbindings bindingsaffinitet til RNA eller DNA 3 , komponentstøkiometrien for nukleinsyre-protein-komplekser 1 og tilvejebringe vigtige nye indsigter om bindingsspecificiteten af ​​RNA-bindingsproteiner via substratkonkurrenceforsøg 1 . Den traditionelle eksperimentelle opsætning for disse analyser består i at blande renset protein med et radioaktivt mærket RNA-substrat. De resulterende komplekser analyseres derefter med ikke-denaturerende (native) polyacrylamidgeler hældt mellem to glasplader efterfulgt af prøveelektroforese i et lodret apparat 3. Selvom denne tilgang er blevet udtømmende anvendt til at tilvejebringe vigtige indsigter i de biokemiske mekanismer, der ligger til grund for bindingen af ​​proteiner til nukleinsyrer, har den også flere begrænsninger. For eksempel har denne grundlæggende strategi relativt lav gennemstrømning, og det er ikke let at tilpasse til applikationer, der kræver analyse af mange bindende reaktioner parallelt. Derudover er det med det traditionelle vertikale apparat udfordrende at potentielt overvåge komplekser ved flere gange under elektroforese 3 , 4 . Her præsenterer vi en tilpasning af EMSA-analysen, der anvender native polyacrylamidgeler, der støbes i et flatbed apparat, vandret elektroforese og fluorescensmærkede RNA-substrater 4 , 5 , 6 , 7 . Indarbejdelsen af ​​disse relativt enkle modifikationer af den basale straTegy giver nogle stærke fordele. Især gør det vandrette flatbed format let at analysere snesevis af prøver samtidigt 4 . For nogle RNA-proteinkomplekser, såsom dem der dannes mellem Bicaudal-C-proteinet og dets RNA-substratelektroforese i en vandret gel, tilvejebringer også en øget evne til at løse forskellige RNA-proteinkomplekser og diskriminere disse fra ubundet RNA-substrat.

<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

Native polyacrylamidgelelektroforese er et grundlæggende værktøj for molekylærbiologi, der er blevet anvendt i vid udstrækning til den biokemiske analyse af RNA-proteininteraktioner. Disse interaktioner er traditionelt analyseret med polyacrylamidgeler frembragt mellem to glasplader og prøver elektroforeseret vertikalt. Polyacrylamidgeler støbes i bakker og elektroforeses horisontalt, men giver flere fordele. For eksempel kan vandrette geler, som anvendes til analyse af komplekser mellem fluorescerende RNA-substrater og specifikke proteiner, afbildes flere gange, idet elektroforese udvikler sig. Dette giver den unikke mulighed for at overvåge RNA-proteinkomplekser på flere punkter under eksperimentet. Derudover gør horisontal gelelektroforese det muligt at analysere mange prøver parallelt. Dette kan i høj grad lette tidskursforsøg samt analysere flere reaktioner samtidigt for at sammenligne forskellige komponenter og betingelser. Her prOvide en detaljeret protokol til generering og anvendelse af horisontal indfødt gelelektroforese til analyse af RNA-Protein-interaktioner.

For at demonstrere kraften og alsidigheden af ​​den horisontale indfødte gelelektroforese analyserede vi bindingen af Xenopus Bicaudal-C (Bicc1) -proteinet til et fluorescensmærket RNA indeholdende et Bicc1-bindingssted. Bicc1-proteiner virker som mRNA-specifikke translationsundertrykkere for at kontrollere beslutninger om cellernes skæbne under moderfasen af ​​dyreudvikling 9 , 10 , <sup class="xre...

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

Native polyacrylamidgelelektroforese er et grundlæggende redskab til analyse af RNA-proteininteraktioner. Traditionelt har de fleste eksperimenter brugt vertikale geler. Imidlertid tilvejebringer vandrette geler flere fordele, såsom muligheden for at overvåge komplekser under elektroforese. Vi leverer en detaljeret protokol til generering og anvendelse af horisontal indfødte gelelektroforese.

Horisontal gelelektroforese til forbedret påvisning af protein-RNA-komplekser

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

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