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[DESCRIPTION]
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<p>This is a sample Mobility Shift Protocol (NFκB). Each oligo labeled with IRDye 700 provided by LI-COR® Biosciences for EMSA reactions will have an optimized protocol to measure the protein-DNA interaction. See the specific EMSA oligo pack insert for more information. As an example, the NFκB protein-DNA interaction will be described in this protocol.</p>
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<p><strong>Please refer to your manual to confirm that this protocol is appropriate for the applications compatible with your Odyssey Imager model.</strong></p>
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[GUIDELINES]
<p><strong>I. Introduction</strong></p>
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<p>Gel shift assays or electrophoretic mobility shift assays (EMSA) provide a simple method to study DNA-protein interactions.This assay is based on the principle that a DNA-protein complex will have different mobility during electrophoresis than non-bound DNA.These shifts can be visualized on a native acrylamide gel using labeled DNA to form the DNA-protein binding complex. To date, protocols require labeling DNA by (1) radioisotope, (2) digoxygenin, or (3) biotin.The Odyssey® Family of Imagers (LI-COR® Biosciences) offers a quick and easily-adapted alternative method to radioisotopic and chemiluminescent detection methods for EMSA analysis and visualization.</p>
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<p>A DNA oligonucleotide end-labeled with LI-COR IRDye is a good substrate for protein binding. LI-COR offers pre-annealed oligonucleotides specific to eight unique binding proteins. DNA detection using IRDye reagents is linear within a 50-fold dilution range, from 9.1 fmol to 0.18 fmol. Additional benefits include no hazardous radioisotope, no gel transfer to membrane or gel drying, no chemiluminescent substrate reagents, and no film exposure. Following electrophoresis, the gel can be imaged while remain- ing in the glass plates. If necessary, the gel can be placed back in the electrophoresis unit and run longer.</p>
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<p>Existing mobility shift assay protocols can be easily transformed into infrared assays by replacing the existing DNA oligonucleotides with oligonucleotides end-labeled with IRDye reagents.The binding conditions and electrophoresis conditions will remain the same as with any other EMSA detection method.</p>
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<p><strong>II. General Methodology</strong></p>
<p><strong>EMSA Oligonucleotides Labeled with IRDye 700</strong></p>
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<p>IRDye 700 p53 Consensus Oligonucleotide</p>
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<p>829-07921</p>
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<p>IRDye 700 STAT3 Consensus Oligonucleotide</p>
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<p>829-07922</p>
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<p>IRDye 700 CREB Consensus Oligonucleotide</p>
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<p>829-07923</p>
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<p>IRDye 700 NFκB Consensus Oligonucleotide</p>
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<p>829-07924</p>
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<p>IRDye 700 AP-1 Consensus Oligonucleotide</p>
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<p>829-07925</p>
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<p>IRDye 700 Sp-1 Consensus Oligonucleotide</p>
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<p>829-07926</p>
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<p>IRDye 700 HIF-1 Consensus Oligonucleotide</p>
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<p>829-07929</p>
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<td style="width: 257px;">EMSA Buffer Kit for the Odyssey</td>
<td style="width: 82px;">829-07910</td>
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<p><strong>Labeling DNA Fragments with IRDye Infrared Dyes</strong></p>
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<p>To obtain DNA fragments end-labeled with IRDye infrared dyes, oligos labeled with IRDye infrared dyes are used. It is critical that the DNA fragment is end-labeled rather than having dye incorporated into the DNA, which interferes with the formation of the DNA-protein complex.</p>
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<p>Oligonucleotides are manufactured in single strand form; therefore, both forward and reverse DNA oligo-nucleotides must be purchased. Once oligonucleotides are obtained, they need to be annealed to form a double-stranded DNA fragment.</p>
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<p>Oligonucleotides are annealed by placing the oligonucleotide set in a 100°C heat block for 5 minutes and then leaving the oligonucleotides in the heat block and turning it off to slowly cool to room temperature.</p>
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<p><em><strong>Important:</strong> Both oligonucleotide sequences should be end-labeled with the same IRDye infrared dye.There is a significant decline (~70%) in signal intensity when using only one end-labeled oligonucleotide.</em></p>
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<p><strong> III. Mobility Shift Sample Protocol (NFκB)</strong></p>
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<p>Each oligo labeled with IRDye 700 provided by LI-COR® Biosciences for EMSA reactions will have an optimized protocol to measure the protein-DNA interaction. See the specific EMSA oligo pack insert for more information. As an example, the NFκB protein-DNA interaction will be described in this protocol.</p>
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<p><strong>See 'STEPS' for protocol. </strong></p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g9ic2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g9ic2w6.png" data-ofn="Screen Shot 2016-12-14 at 10.13.46 AM.png" /></p>
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<p>One of the benefits of using the Odyssey® Infrared Imaging System for EMSA analysis is that it provides an easy method for quantification. However, there are issues to consider when using the Odyssey Imager to quantify EMSA results.The primary issue is that the free DNA fragment has much less signal than the DNA fragment when bound to a protein, making quantification of the unbound DNA inaccurate.The addition of DTT/Tween® 20 to the binding reaction stabilizes the dye and reduces this phenomenon. In addition, it is unrealistic to perform quantification analyses under the assumption that the free DNA band in the control, containing DNA only (no extract), should equal the sum of the signals of the free and bound DNA in the samples where the protein-DNA binding reaction occurs. Using end-labeled oligonucleotide duplexes as the DNA source and nuclear extract as a protein source renders this assumption impractical, due to the non-specific binding that occurs from using a nuclear extract. Oligonucleotides can also complicate quantification because the free oligonucleotides form a smear rather than a tight band.This makes it more difficult to assign an intensity value to bands.</p>
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<p><strong>Optimization</strong></p>
<p><strong>Binding Reaction</strong></p>
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<p>A universal binding condition that applies to every protein-DNA interaction cannot be recommended, since binding conditions are specific for each protein-DNA interaction.Thus, the user should establish binding reaction conditions for each protein-DNA pair. Binding buffer should be the same for this method as with any other mobility shift detection method used.</p>
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<p>After the addition of DNA to the protein-buffer mix, reactions are incubated to allow protein to bind to DNA. Time required for binding is the same as when radioactively-labeled DNA fragments are used; a typical incu- bation condition is 20-30 minutes at room temperature. Since IRDye reagents are sensitive to light, it is best to keep binding reactions in darkness during incubation periods (e.g., put tubes into a drawer or simply cover the tube rack with aluminum foil). After the incubation period, native loading dye is added to the binding reaction.</p>
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<p><em>NOTE: In some cases, it was observed that DNA control reactions (no protein) have lower signal than reactions containing protein.This may be due to lower stability of the dye in certain buffer conditions.The addition of 5 mM DTT and 0.5%Tween 20 to all reactions reduces this phenomenon.</em></p>
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<p><em>IMPORTANT: It is critical not to use any blue loading dye (e.g., bromophenol blue), as this will be visible on the Odyssey® image. Use 10X Orange loading dye instead (LI-COR®, P/N 927-10100).</em></p>
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<p><em><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g9jc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g9jc2w6.png" data-ofn="Screen Shot 2016-12-14 at 10.16.42 AM.png" /></em></p>
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<p><em><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g9kc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g9kc2w6.png" data-ofn="Screen Shot 2016-12-14 at 10.17.15 AM.png" /></em></p>
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<p>Competition using mutant DNA duplexes is another common method to determine binding specificity. A mutant DNA sequence is used to compete with the wild-type binding sequence. Specific binding is observed when mutant DNA (unlabeled) does not reduce the binding of labeled wild-type DNA.Two-color analysis of mutant vs. wild-type binding is done using the Odyssey Infrared Imaging System.The wild-type oligos are labeled with IRDye 700 phosphoramidite and mutant oligos with IRDye 800 phosphoramidite. In the figure above, the mutant non-specific binding is very intense (800 nm image); however, there is no decrease in wild-type binding (700 nm image).</p>
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<p>Free IRDye 700 AP-1 consensus oligonucleotide and IRDye 800 AP-1 mutant oligonucleotide with no nuclear extract;</p>
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<p>Nuclear extract with 0:1 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1 mutant oligonucleotide;</p>
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<p>Nuclear extract with 1:0 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1 mutant oligonucleotide;</p>
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<p>Nuclear extract with 1:1 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1 mutant oligonucleotide;</p>
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<p>Nuclear extract with 1:2 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1 mutant oligonucleotide;</p>
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<p>Nuclear extract with 1:3 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1 mutant oligonucleotide;</p>
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<p>Nuclear extract with 1:4 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1 mutant oligonucleotide;</p>
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<p>Nuclear extract with 1:5 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1 mutant oligonucleotide;</p>
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<p><strong>References</strong></p>
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<p>1. Wolf, S.S., Hopley, J.G., and Schweizer, M. (1994) The Application of 33P-Labeling in the Electrophoretic Mobility Shift Assay. Biotechniques <strong>16</strong>, 590-592.</p>
<p>2. Suske, G., Gross, B., and Beato, M. (1989) Non-radioactive method to visualize specific DNA-protein interactions in the band shift assay. Nucleic Acids Research, <strong>17</strong>, 4405.</p>
<p>3. Ludwig, L.B., Hughes, B.J., and Schwartz, S.A. (1995) Biotinylated probes in the electrophoretic mobility shift assay to examine specific dsDNA, ssDNA or RNA-protein interactions. Nucleic Acids Research, <strong>23</strong>, 3792-3793.</p>
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