Electrophoretic Mobility Shift

Electrophoretic Mobility Shift®Assay (EMSA) Using IRDyeOligonucleotidesDeveloped for:Odyssey®Family of ImagersPlease refer to your manual to confirm

that this protocol is appropriate for the

applications compatible with your

Odyssey Imager hed May 2004. Revised October 2011.

The most recent version of this protocol

is posted at

Page 2 –EMSA Using IRDye®OligonucleotidesTable of UCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 2GENERAL METHODOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 2EMSA Oligonucleotides Labeled with IRDye®700. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 2Labeling DNA Fragments with IRDye Infrared Dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . Page 3MOBILITY SHIFT SAMPLE PROTOCOL (NFκB). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 3Optimization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page nces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 6I. IntroductionGel 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 differentmobility during electrophoresis than non-bound DNA. These shifts can be visualized on a native acryla-mide gel using labeled DNA to form the DNA-protein binding complex. To date, protocols require label-ing 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 chemiluminescentdetection methods for EMSA analysis and visualization.

A DNA oligonucleotide end-labeled with LI-COR IRDye is a good substrate for protein binding. LI-CORoffers pre-annealed oligonucleotides specific to eight unique binding proteins. DNA detection usingIRDye reagents is linear within a 50-fold dilution range, from 9.1 fmol to 0.18 fmol. Additional benefitsinclude 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.

Existing mobility shift assay protocols can be easily transformed into infrared assays by replacing theexisting DNA oligonucleotides with oligonucleotides end-labeled with IRDye reagents. The binding con-ditions and electrophoresis conditions will remain the same as with any other EMSA detection method.

II. General MethodologyEMSA Oligonucleotides Labeled with IRDye 700Part NumberIRDye 700 p53 Consensus Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . 829-07921IRDye 700 STAT3 Consensus Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . 829-07922IRDye 700 CREB Consensus Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . 829-07923IRDye 700 NFκB Consensus Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . 829-07924IRDye 700 AP-1 Consensus Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . 829-07925IRDye 700 Sp-1 Consensus Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . 829-07926IRDye 700 HIF-1 Consensus Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . 829-07929IRDye700 ARE (Androgen Receptor) Consensus Oligonucleotide. . . . . . . . . 829-07933EMSA Buffer Kit for the Odyssey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829-07910

EMSA Using IRDye®Oligonucleotides –Page 3Labeling DNA Fragments with IRDye Infrared DyesTo obtain DNA fragments end-labeled with IRDye infrared dyes, oligos labeled with IRDye infrared dyes areused. 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.

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 adouble-stranded DNA ucleotides are annealed by placing the oligonucleotide set in a 100°C heat block for 5 minutes andthen leaving the oligonucleotides in the heat block and turning it off to slowly cool to room ant:Both oligonucleotide sequences should be end-labeled with the same IRDye infrared dye. Thereis a significant decline (~70%) in signal intensity when using only one end-labeled . Mobility Shift Sample Protocol (NFκB)Each oligo labeled with IRDye 700 provided by LI-COR®Biosciences for EMSA reactions will have an opti-mized protocol to measure the protein-DNA interaction. See the specific EMSA oligo pack insert for moreinformation. As an example, the NFκB protein-DNA interaction will be described in this document.

Gel Preparation:Native pre-cast polyacrylamide gels such as 5% TBE (BioRad) or 4-12% TBE (Invitrogen)are recommended. Alternatively, the recipe below can be used to prepare a 4% native gel.

NOTE:The protein shift detected on each gel type (i.e., 5% vs 4-12%) will be e4% native polyacrylamide gel containing 50 mM Tris, pH 7.5; 0.38 M glycine; and 2 mM EDTA:For 40 mL mix:5 mL 40% polyacrylamide stock (Polyacrylamide-BIS ratio = 29:1)2 mL 1 M Tris, pH 7.57.6 mL 1 M Glycine160 μL 0.5 M EDTA26 mL H2O200 μL 10% APS30 μL TEMEDPour the gel between glass plates and wait about 1-2 hours to Preparation: EMSA oligonucleotides from LI-COR Biosciences are oligos in 1X TE for final concentration of 20 pmol/μ 5 μL of forward IRDye 700 oligo into a new tube and add 5 μL of reverse IRDye 700 oligos by placing the oligo set in a 100°C heat block for 3 minutes. Leave the oligos in

the heat block and turn it off to slowly cool to room annealed oligos 1 μL in 199 μL water. This is your working DNA stock. Oligos can be

stored at -20°C for up to a year if protected from g Reaction: For NFκB IRDye 700 oligonucleotide, the following binding reaction is a good

starting point.

µLReaction10X Binding Buffer (100 mM Tris, 500 mM KCl, 10 mM DTT; pH 7.5) 2Poly(dI•dC) 1 μg/μL in 10 mM Tris, 1 mM EDTA; pH 7.5 125 mM DTT/2.5% Tween®20 2Water 13IRDye 700 NFκB 1Raji nuclear extract (Positive control) (5 μg/μL) 1TOTAL 20

Page 4 –EMSA Using IRDye®OligonucleotidesAfter the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding toDNA. A typical incubation condition is 20-30 minutes at room temperature. Since IRDye 700 infrared dye issensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put tubesinto a drawer or cover the tube rack with aluminum foil).

Electrophoresis: 1 μL of 10X Orange loading dye (LI-COR®, P/N 927-10100), mix, and load on a the gel at 10 V/cm for about 30 minutes in non-denaturing buffer (i.e., 1X TGE or TBE buffer).NOTE:For best results, electrophoresis should be performed in the dark (simply put a cardboard box

over the electrophoresis apparatus).Imaging:Gels can be imaged either inside the glass plates or removed from the glass plate. When

removing gel from the glass plates, take care not to deform or tear the gel. Scan the gel. Please refer

to your manual for specific information on your model of imager.

Figure 1. IRDye 700 NFκB oligonucleotides were separatedon a native polyacrylamide gel (4-12% TBE, InvitrogenEC62352BOX) and imaged on the Odyssey®InfraredImaging System.

Lane 1) no nuclear extract;

Lanes 2 and 5) 10 μg Raji nuclear extract;

Lanes 3 and 6) 5 μg Raji nuclear extract;

Lanes 4 and 7) 2.5 μg Raji nuclear 2. The uppermost shifted band inLanes 2-7 of Figure 1 was analyzed todetermine the level of NFkB binding to

the IRDye 700 NFκB ityµg Raji nuclear extract

One of the benefits of using the Odyssey®Infrared Imaging System for EMSA analysis is that it provides aneasy method for quantification. However, there are issues to consider when using the Odyssey Imager toquantify EMSA results. The primary issue is that the free DNA fragment has much less signal than the DNAfragment when bound to a protein, making quantification of the unbound DNA inaccurate. The addition ofDTT/Tween®

20 to the binding reaction stabilizes the dye and reduces this phenomenon. In addition, it is un-realistic to perform quantification analyses under the assumption that the free DNA band in the control, con-taining DNA only (no extract), should equal the sum of the signals of the free and bound DNA in the sampleswhere the protein-DNA binding reaction occurs. Using end-labeled oligonucleotide duplexes as the DNAsource and nuclear extract as a protein source renders this assumption impractical, due to the non-specificbinding that occurs from using a nuclear extract. Oligonucleotides can also complicate quantification becausethe free oligonucleotides form a smear rather than a tight band. This makes it more difficult to assign anintensity value to bands.

EMSA Using IRDye®Oligonucleotides –Page 5OptimizationBinding ReactionA universal binding condition that applies to every protein-DNA interaction cannot be recommended, sincebinding 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 the addition of DNA to the protein-buffer mix, reactions are incubated to allow protein to bind to 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 bestto keep binding reactions in darkness during incubation periods (e.g., put tubes into a drawer or simply coverthe tube rack with aluminum foil). After the incubation period, native loading dye is added to the : 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 ANT: 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).HeLa-2

hr

SRHeLa-4

hr

SRHeLaFigure 3. AP-1 EMSA using IRDye 700 end-labeledoligonucleotide duplex.

It is common to use unlabeled DNA duplex to deter-mine binding specificity. Excess unlabeled DNA isadded to the binding reaction; therefore, it competeswith the labeled DNA for binding sites. If competitioneliminates labeled DNA binding, no shift is observed(see last three lanes in gel), indicating that the bindingreaction is ition reactions contained 100-fold molar excessof wild-type oligonucleotide duplex. Nuclear extracts ofHeLa, HeLa 2-hour serum response, and HeLa 4-hourserum response, were used to visualize an increase

in AP-1 binding as a result of the serum response treat-ment to the HeLa cells.–+ + + + + +Nuclear Extract

IRDye 700 AP-1 oligo+ + + + + + +–––+ + +AP-1 wild-type competitor oligo–HeLa-2

hr

SRHeLa-4

hr

SRNo

ExtractHeLa

Page 6 –EMSA Using IRDye®OligonucleotidesAP-1 consensus/mutant binding700 nm/800 nm imageAP-1 consensus/mutant binding700 nm imageAP-1 consensus/mutant binding800 nm imageFigure 4. AP-1 EMSA using 2.5 μg HeLa 4-hour serum response nuclear extract to demonstratebinding specificity of AP-1 consensus DNA duplex. Binding specificity determination usingOdyssey®two-color ition using mutant DNA duplexes is another common method to determine binding specificity. Amutant DNA sequence is used to compete with the wild-type binding sequence. Specific binding is observedwhen mutant DNA (unlabeled) does not reduce the binding of labeled wild-type DNA. Two-color analysis ofmutant vs. wild-type binding is done using the Odyssey Infrared Imaging System. The wild-type oligos arelabeled with IRDye 700 phosphoramidite and mutant oligos with IRDye 800 phosphoramidite. In the figureabove, the mutant non-specific binding is very intense (800 nm image); however, there is no decrease inwild-type binding (700 nm image).

Lane 1–Free IRDye 700 AP-1 consensus oligonucleotide and IRDye 800 AP-1 mutant oligonucleotide

with no nuclear extract;Lane 2–Nuclear extract with 0:1 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1

mutant oligonucleotide;Lane 3–Nuclear extract with 1:0 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1

mutant oligonucleotide;Lane 4–Nuclear extract with 1:1 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1mutantoligonucleotide;Lane 5–Nuclear extract with 1:2 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1mutantoligonucleotide;Lane 6–Nuclear extract with 1:3 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1mutant oligonucleotide;Lane 7–Nuclear extract with 1:4 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1

mutant oligonucleotide;Lane 8–Nuclear extract with 1:5 ratio of IRDye 700 AP-1 consensus oligonucleotide to IRDye 800 AP-1mutant oligonucleotide;References1. Wolf, S.S., Hopley, J.G., and Schweizer, M. (1994) The Application of

33P-Labeling in the Electrophoretic

Mobility Shift Assay. Biotechniques16, 590-592.2. Suske, G., Gross, B., and Beato, M. (1989) Non-radioactive method to visualize specific DNA-proteininteractions in the band shift assay. Nucleic Acids Research, 17, 4405.3. 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, 23, 3792-3793.

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