Name: an optimized protocol for electrophoretic mobility shift assay using infrared fluorescent dye-labeled oligonucleotides
Uploaded: 2016-11-29T13:00:00
Description: Watch this Scientific Journal Video about An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides at JoVE.com

This work was supported by an Alfred P. Sloan Research Fellowship (to C.-F.C.) and an NIH R01 grant (5R01GM098026-05 to C.-F.C.). We thank David Crowe for access to the advanced infrared imaging system.

NOTE: EMSAs using fluorescently labeled DNA probes or other forms of labeled DNA share the same protocols for protein or cell extract preparation, protein-DNA binding reaction, and PAGE gel preparation and running (Figure 1A). The key differences are DNA labeling procedures, post-run gel processing steps, and detection methods.
1. Gel Preparation
<ol>
	<li>Prepare 5% native polyacrylamide gel containing 0.5x&#160;TBE (45 mM Tris-Borate, 1 mM EDTA) and 2.5% glycerol, using a mini protein gel syst...

<ol>
	<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, 1849-1861 (2007).</li><li>Ludwig, L. B., Hughes, B. J., Schwartz, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylated+probes+in+the+electrophoretic+mobility+shift+assay+to+examine+specific+dsDNA,+ssDNA+or+RNA-protein+interactions.">Biotinylated probes in the electrophoretic mobility shift assay to examine specific dsDNA, ssDNA or RNA-protein interactions.</a> Nucleic Acids Res. 23, 3792-3793 (1995).</li><li>Engler-Blum, G., Meier, M., Frank, J., Muller, G. 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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=LUEGO:+a+cost+and+time+saving+gel+shift+procedure.">LUEGO: a cost and time saving gel shift procedure.</a> Biotechniques. 51, 267-269 (2011).</li><li>Poulin-Laprade, D., Burrus, V. <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+Using+Radiolabeled+DNA+Probes.">Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes.</a> Methods Mol Biol. 1334, 1-15 (2015).</li><li>Ruscher, 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=A+fluorescence+based+non-radioactive+electrophoretic+mobility+shift+assay.">A fluorescence based non-radioactive electrophoretic mobility shift assay.</a> J Biotechnol. 78, 163-170 (2000).</li><li>Pagano, J. 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fEMSAs are an efficient tool to investigate protein-DNA interactions, and are an alternative to radioactive labeling of DNA. Fluorescent dyes such as infrared dyes are commercially available, and provide a safer and more environmentally friendly method compared to radioactive DNA labeling. EMSAs using infrared fluorescent dye-labeled oligonucleotides do not require postrun gel processing steps, and therefore save time and cost compared to other DNA labeling techniques. The time to detect bands using radioactive EMSAs can...

EMSAs are used to analyze interactions between DNA and proteins by using native PolyAcrylamide Gel Electrophoresis (PAGE) to resolve a mixture of a protein of interest and a labeled DNA probe containing potential target sites of the protein1. A DNA probe bound with protein will migrate slower compared with a free DNA probe, and is therefore retarded in its migration through a polyacrylamide matrix. Radiolabeling of DNA by 32P has been the predominant method for detection in EMSAs. Although the application of radiolabeling in biochemical research has been beneficial, methods of alternative DNA labeling with comparable sensitivity are being employed due to the health and safety risks associated with handling radioactivity. These alternative methods include conjugation of DNA with biotin2 or digoxigenin (DIG)3 (both of which are then detected by chemiluminescent systems), SYBR green staining of the PAGE gels4, or direct detection of DNA-fluorescent dye conjugates by scanning the gel5,6.
The resolved gels of EMSAs using radioactively labeled DNA probes require postrun processing through autoradiography films or a phosphorimager system to detect radioactive signals1,7. Gels of EMSAs using biotin-2 or DIG-conjugated3 DNA probes must also be processed and transferred onto a suitable membrane and then detected by chemiluminescence6. SYBR green staining of the gel requires postrun gel staining and a fluorescent scanner4. Since postrun gel processing steps are required for EMSAs using these DNA labeling techniques, the resolved gel can be assayed only once. In contrast, DNA probes labeled with fluorophores can be directly detected in the gel inside the glass plates by a scanner. Therefore, the DNA-protein interactions can be monitored and assayed several times at different time points during the run, which significantly reduces time and cost. The DNA-fluorescent dye conjugates that have been used for EMSAs include Cy36,8, Cy56,8, fluorescein9, and infrared fluorescent dyes4-6.
Transcriptional regulation requires protein-DNA interactions of transcription factors and their target genes. Coordination of these interactions generates diverse cell types from a common progenitor during animal development. An unbiased forward genetic screen identified a missense mutation, G73E, in the highly conserved HMG DNA-binding domain of the Caenorhabditis elegans transcription factor SOX-2. The mutation results in a cell identity transformation of AWB olfactory neurons into AWC olfactory neurons at molecular, morphological, and functional levels5,10. SOX-2 differentially regulates the terminal differentiation of AWB and AWC neurons by interacting with context-dependent partner transcription factors and respective DNA target sites5,10. SOX-2 partners with LIM-4 (LHX) in terminal AWB neuronal differentiation, while SOX-2 partners with CEH-36 (OTX/OTD) in terminal AWC neuronal differentiation. Luciferase reporter assays show that SOX-2 and mutant SOX-2G73E proteins have cooperative interactions with transcriptional cofactors LIM-4 and CEH-36 to activate a promoter expressed in AWB and AWC neurons. However, SOX-2 and mutant SOX-2G73E displayed differential activation properties of the promoter. To investigate the molecular basis of differential DNA binding activities of SOX-2 and SOX-2G73E, fEMSAs were performed with these proteins and their potential target sites.
First, a bioinformatics approach was taken to identify the biologically relevant DNA binding sequences within the 1 kb promoter region used in the luciferase assay. Since multiple potential SOX-2 binding sites were present throughout the promoter, we focused on predicted SOX-2 binding sites adjacent to putative CEH-36 or LIM-4 binding sites and evolutionarily conserved between various nematode species. These sequences were deleted or mutated, and subsequently tested in vivo for their activity to express GFP reporter transgenes in AWB or AWC neurons. Through this approach, we identified potential LIM-4/SOX-2 and CEH-36/SOX-2 adjacent target sites that are specifically required for the expression of GFP in AWB and AWC neurons, respectively5. We investigated the potential differences in the DNA binding of SOX-2 and SOX-2G73E using fEMSA with the identified LIM-4/SOX-2 and CEH-36/SOX-2 adjacent target sites. Our EMSA analysis showed that SOX-2G73E did not bind the DNA probe containing the LIM-4/SOX-2 adjacent target sites (required for gene expression in AWB) as efficiently as WT SOX-2 did. However, SOX-2 and SOX-2G73E had no difference in binding the DNA probe containing the CEH-36/SOX-2 adjacent target site (required for gene expression in AWC)5,10. Our fEMSA analyses provide mechanistic insight into the nature of the SOX-2G73E mutation in affecting specific DNA binding activity for the AWB-to-AWC cell identity transformation phenotype. Here, we describe an optimized protocol of fEMSA using purified 6xHis-SOX-2 or 6xHis-SOX-2G73E together with infrared fluorescent dye-labeled DNA probes containing the LIM-4/SOX-2 adjacent target sites as a case study to tackle an important biological question.

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			<td height="21" style="height:21px;width:332px;">30% Acrylamide/Bis Solution, 37.5:1&#160;</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:207px;">1610158</td>
			<td style="width:281px;">Acrylamide is harmful and toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">6x-His Epitope Tag Antibody (HIS.H8)</td>
			<td style="width:199px;">ThermoFisher</td>
			<td style="width:207px;">MA1-21315</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti Flag M2 antibody&#160;</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:207px;">F3165-.2MG</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Bovine Serum Albumin molecular-biology-grade</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:207px;">B9000S</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:332px;">5&#39;IRDye700-labeled DNA Oligos</td>
			<td style="width:199px;">Integrated DNA Technologies</td>
			<td style="width:207px;">Custom DNA oligo</td>
			<td style="width:281px;">These are referred as 5&#39;Dye-labeled or infrared fluorescent dye-labeled DNA oligos in the manuscript. The company will custom synthesize 5&#39; IRDye labeled DNA oligonucleotides.&#160; Requires minimum 100&#956;M scale synthesis and HPLC purification.</td>
		</tr>
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			<td height="21" style="height:21px;width:332px;">Klenow Fragment (3&#39;--&#62;5&#39; exo-)</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:207px;">M0212S</td>
			<td style="width:281px;"></td>
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			<td height="21" style="height:21px;width:332px;">LightShif Poly (dI-dC)</td>
			<td style="width:199px;">ThermoFisher</td>
			<td style="width:207px;">20148E</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:332px;">Mini-PROTEAN&#160;Vertical Electrophoresis Cell&#160;</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:207px;">1658000FC&#160;</td>
			<td style="width:281px;">This is referred as a mini protein gel system in the manuscript. Any electrophoretic system can be used as long as they are clear glass plates of less than 25cm x 25cm in size.</td>
		</tr>
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			<td height="42" style="height:42px;width:332px;">Odyssey CLx Infrared Imaging System</td>
			<td style="width:199px;">LI-COR Biotechnology</td>
			<td style="width:207px;">Odyssey CLx Infrared Imagng System</td>
			<td style="width:281px;">This is referred as an advanced infrared imaging system in the mansucript.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Odyssey Fc Imaging System&#160;</td>
			<td style="width:199px;">LI-COR Biotechnology</td>
			<td style="width:207px;">Odyssey Fc Dual-Mode Imaging System</td>
			<td style="width:281px;">This is referred as a near-infrared fluorescent imaging system primarily for Western blots in the mansucript.</td>
		</tr>
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			<td height="42" style="height:42px;width:332px;">Image Studio software (version 4.0)</td>
			<td style="width:199px;">LI-COR Biotechnology</td>
			<td style="width:207px;">Image Studio software&#160;</td>
			<td style="width:281px;">This is referred as a particular imaging software in the mansucript.&#160;</td>
		</tr>
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			<td height="21" style="height:21px;width:332px;">Orange G</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:207px;">O3756-25G</td>
			<td style="width:281px;"></td>
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			<td height="21" style="height:21px;width:332px;">6x Orange loading dye</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td>0.25% Orange G; 30% Glycerol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5x TBE</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td>45 mM Tris-Borate; 1 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1x TE</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td>10 mM Tris-HCl; 1 mM EDTA, pH8.0</td>
		</tr>
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			<td height="42" style="height:42px;width:332px;">1x STE</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td style="width:281px;">100 mM NaCl; 10 mM Tris-HCl, pH8.0; 1 mM EDTA</td>
		</tr>
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			<td height="63" style="height:63px;">5x Binding Buffer</td>
			<td style="width:199px;"></td>
			<td style="width:207px;"></td>
			<td style="width:281px;">50 mM Tris-HCl, pH7.5; 50 mM NaCl; 200 mM KCl; 5 mM MgCl2; 5 mM EDTA, pH8.0; 5 mM DTT; 250 ug/ml BSA</td>
		</tr>
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</table>

an optimized protocol for electrophoretic mobility shift assay using infrared fluorescent dye-labeled oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

Orange G loading dye (6x: 0.12 g Orange G in 100 mL&#160;30% Glycerol) can be added to the binding reaction prior to loading to visualize the progress of the electrophoresis. Other loading dyes including bromophenol blue will be detected during scanning and therefore interfere with image analysis (Figure 1B).
In some instances of EMSAs, especially if nuclear extract preparation is used, poly deoxyinosinic-deoxyc...

Watch this Scientific Journal Video about An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides at JoVE.com

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

The overall goal of this assay is to detect protein-DNA interactions using non-radioactive probes. This method can help answer key questions in the molecular biology and biochemistry fields, such as determining DNA target sequence of proteins. The main advantage of this technique is that it is an easy, safe, and time saving alternative to using radioactive probes.To begin this procedure, prepare a five percent native polyacrylamide gel containing 0.5x tris borate EDTA or TBE, and 2.5%glycerol using a mini protein gel system. To prepare 30 milliliters of gel solution for four gels, mix distilled water, TBE, ammonium persulfate, TEMED, acrylamide bis, and glycerol. Pass the gel solution immediately.After polymerization, wrap the gels in clear plastic wrap pre-wetted with 0.5x TBE and store them at four degrees Celsius. Next, design a long oligonucleotide for approximately 51 mers. Design complementary short oligonucleotides for approximately 14 mers with infrared fluorescent dye modification at the five prime terminus.Once the oligonucleotides have been prepared, resuspend them in 1x Tris-EDTA buffer to a final concentration of 100 micromolar. For annealing, mix 0.6 microliters of five prime dye short oligonucleotide, 1.2 microliters of long oligonucleotide, and 28.2 microliters of sodium chloride Tris-EDTA buffer in a 1.5 milliliter tube. Place the tube in boiling water for five minutes, then turn off the heat source and allow the water with the annealed oligonucleotides to cool overnight in the dark.To make double-stranded DNA probes, mix 30 microliters of the annealed oligonucleotides with DNTPs, Klenow buffer, Klenow five prime dye tag, and double distilled water in a 0.2 milliliter PCR tube. Incubate the mixture for 60 minutes at 37 degrees Celsius in a PCR machine. Then, add 3.4 microliters of 0.5 molar EDTA to stop the reaction, and heat inactivate at 75 degrees Celsius for 20 minutes in the PCR machine.Following this, dilute the filled in probes with sodium chloride Tris-EDTA buffer to a final concentration of 0.1 micromolar. Store the oligonucleotides at minus 20 degrees Celsius in the dark until ready to use. To prepare unlabeled probes, mix 20 microliters of long oligonucleotide, 20 microliters of Long R oligonucleotide, and 60 microliters of sodium chloride Tris-EDTA buffer in a 1.5 milliliter tube.Place the tube in boiling water for five minutes, then turn off the heat source and allow the water with the annealed oligos to cool overnight. Prepare one milliliter of 5x binding buffer by mixing Tris HCl, Sodium Chloride, Potassium Chloride, Magnesium Chloride, EDTA, DTT, BSA, and double-distilled water. Prior to setting up the binding reactions, pre-run the 5%native polyacrylamide gel in 0.5x TBE and 2.5%glycerol to remove all traces of ammonium persulfate at 80 volts for 30 minutes to one hour, or until the current no longer varies with time.Next, mix four microliters of 5x binding buffer, 80 to 200 nanograms of purified protein A, one microliter of 0.1 micromolar dye conjugated probe, and double-distilled water. Incubate the mixture at room temperature in the dark for 15 minutes. Following incubation, load all of the binding reaction onto the gel and run the gel at 10 volts per centimeter to the desired distance.Cover the gel apparatus with aluminum to keep the gel in the dark as much as possible. Clean the scanner bed of an infrared imaging system with distilled water. Wipe dry the glass plates containing the gel and place them on the scanner bed.Open the infrared imaging software and click on the Acquire tab. For thicker plates, use the settings of 700 for channel, auto for intensity, 84 micrometers for resolution, medium for quality, and 3.5 millimeters for focus offset. Select the area that the gel occupies on the scanner.Finally, click Start to begin the scan. The progress of electrophoresis can be visualized with Orange G loading dye, whereas Bromophenol blue is detected during scanning and therefore interferes with image analysis. Addition of dI-dC to the binding reaction abolished the binding of purified 6xHis-SOX-2 with the five prime dye DNA probes.The LIM-4 mutant and SOX-2 cold probe mutated in the LIM-4 binding site is as efficient as the wild type cold probe in competing with the infrared fluorescent dye labeled probe for binding to 6xHis-SOX-2. However, the competition efficiency of the LIM-4 and SOX-2 mutant cold probe mutated in the SOX-2 binding site is much lower than the wild type cold probe for binding to 6xHis-SOX-2. Supershift assays of the SOX-2 DNA complex using 6xHis and flag epitopes resulted in a supershifted SOX-2 DNA band.Once mastered, this technique can be done in two to four hours if it is performed properly. While attempting this procedure, it is important to remember to keep the infrared probes in the dark. Following this procedure, other methods like CRISPR cas9 genome editing can be performed in order to answer additional questions like the importance of a particular DNA binding sequence.After its development, this technique paved the way for researchers in the field of biochemistry to explore protein-DNA interactions in various model organisms. After watching this video, you should have a good understanding of how to determine protein-DNA interactions using non-radioactive DNA labels. Don't forget that working with acrylamide can be extremely hazardous and precautions such as personal protective equipment should always be taken while performing this procedure.

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