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>
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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>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		<tr height="42">
			<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>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		<tr height="42">
			<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>
		<tr height="63">
			<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>
	</tbody>
</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. ...

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