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

We thank Laura Vanderploeg for preparing figures. Work in the Sheets lab is supported by NSF grant 1050395 and NIH grant (R21HD076828). Work in the Ryder lab is supported by NIH grants R01GM117237 and R01GM117008. Megan Dowdle is supported by a SciMed GRS Advanced Opportunity Fellowship through University of Wisconsin-Madison Graduate School and Biotechnology Training Program through the National Institute of General Medical Sciences of the National Institutes of Health (T32GM008349).

1. Preparation of the Horizontal Native Polyacrylamide Gel4.
<ol>
	<li>Preparation of the materials needed.
	<ol>
		<li>For the horizontal gel apparatus, use a gel box (37 cm x 24 cm) with a 27 cm x 21 cm tray and a capacity for two 24-well combs. This setup provides a total of 48 samples that can be analyzed simultaneously.</li>
		<li>Prepare the following reagents: 40% Acrylamide-Bis 19:1, 5x TBE (Tris-Borate-EDTA pH 8.0), TEMED, and 10% APS (ammonium persulfate).</li>
	</ol>
...

<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nat Protoc. 2 (8), 1849-1861 (2007).</li><li>Pagano, J. M., Farley, B. M., Essien, K. I., Ryder, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+recognition+by+the+embryonic+cell+fate+determinant+and+germline+totipotency+factor+MEX-3.">RNA recognition by the embryonic cell fate determinant and germline totipotency factor MEX-3.</a> Proc Natl Acad Sci USA. 106 (48), 20252-20257 (2009).</li><li>Royer, C. A., Scarlata, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+approaches+to+quantifying+biomolecular+interactions.">Fluorescence approaches to quantifying biomolecular interactions.</a> Meth Enzymol. 450, 79-106 (2008).</li><li>Su, C., Wang, F., Ciolek, D., Pan, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoresis+of+proteins+and+protein-protein+complexes+in+native+polyacrylamide+gels+using+a+horizontal+gel+apparatus.">Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.</a> Anal Biochem. 223, 93-98 (1994).</li><li>Buczek, P., Horvath, M. 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. M., Clingman, C. C., Ryder, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+approaches+to+monitor+protein-nucleic+acid+interactions+using+fluorescent+probes.">Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes.</a> RNA. 17 (1), 14-20 (2011).</li><li>Foley, T. L., 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=Platform+to+Enable+the+Pharmacological+Profiling+of+Small+Molecules+in+Gel-Based+Electrophoretic+Mobility+Shift+Assays.">Platform to Enable the Pharmacological Profiling of Small Molecules in Gel-Based Electrophoretic Mobility Shift Assays.</a> J Biomol Screen. 21 (10), 1125-1131 (2016).</li></ol>

Native polyacrylamide gels are an invaluable tool for investigating protein-RNA interactions and traditionally these gels are electrophoresed vertically2,3. We have used a modification of the protocol that substitutes native polyacrylamide gels created and electrophoresed horizontally1,4,6,7,15. These ...

Electrophoretic mobility shift assays (EMSAs) have proven to be an invaluable biochemical tool to analyze specific protein-nucleic acid interactions1,2,3. These assays can provide important information regarding the binding affinity of proteins to RNA or DNA3, the component stoichiometry of nucleic acid-protein complexes1 and provide important new insights about the binding specificity of RNA binding proteins via substrate competition experiments1.
The traditional experimental setup for these assays consists of mixing purified protein with a radiolabeled RNA substrate. The resulting complexes are then analyzed with non-denaturing (native) polyacrylamide gels poured between two glass plates followed by sample electrophoresis in a vertical apparatus3. While this approach has been used exhaustively to provide important insights in the biochemical mechanisms that underlie the binding of proteins to nucleic acids, it also has several limitations. For example, this basic strategy has relatively low throughput and it is not readily adaptable for applications that require analyzing many binding reactions in parallel. In addition, with the traditional vertical apparatus it is challenging to potentially monitor complexes at multiple times during electrophoresis3,4.
Here we present an adaptation of the EMSA assay that uses native polyacrylamide gels cast in a flatbed apparatus, horizontal electrophoresis and fluorescently labeled RNA substrates4,5,6,7. The incorporation of these relatively simple modifications to the basic strategy provides some powerful advantages. In particular, the horizontal flatbed format easily lends itself to analyzing dozens of samples simultaneously4. Also, for some RNA-protein complexes, such as those formed between the Bicaudal-C protein and its RNA substrate electrophoresis in a horizontal gel provides an increased ability to resolve distinct RNA-protein complexes and discriminate these from unbound RNA substrate.

<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 polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

To demonstrate the power and versatility of the horizontal native gel electrophoresis we analyzed the binding of the Xenopus Bicaudal-C (Bicc1) protein to a fluorescently labeled RNA containing a Bicc1 binding site. Bicc1 proteins function as mRNA-specific translational repressors to control cell fate decisions during the maternal stages of animal development9,10,11,<sup class...

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 polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

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

Research

JoVE Journal

Biochemistry

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

Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

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

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

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

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

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

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

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

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

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

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

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