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Sample Protocol

Mobility Shift Sample Protocol (NFκB)

IRDye® 700 oligos include an optimized protocol to measure the protein:DNA interaction. See the specific EMSA oligo pack insert for more information.

  1. 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 unique.

    Prepare 4% native polyacrylamide gel containing 50 mM Tris, pH 7.5; 0.38 M glycine; and 2 mM EDTA:

  2. For 40 mL mix:

    • 5 mL 40% polyacrylamide stock (Polyacrylamide-BIS ratio = 29:1)
    • 2 mL 1 M Tris, pH 7.5
    • 7.6 mL 1 M Glycine
    • 160 μL 0.5 M EDTA
    • 26 mL H2O
    • 200 μL 10% APS
    • 30 μL TEMED
    • Pour the gel between glass plates and wait about 1-2 hours to polymerize.

  3. Oligo Preparation:

    EMSA oligonucleotides from LI-COR Biosciences are pre-annealed.

    • Dilute oligos in 1X TE for final concentration of 20 pmol/μL.
    • Place 5 μL of forward IRDye 700 oligo into a new tube and add 5 μL of reverse IRDye 700 oligo.
    • Anneal 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 temperature.
    • Dilute 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 light.
  4. Binding Reaction:

    For NFκB IRDye 700 oligonucleotide, the following binding reaction is a good starting point.

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

    After the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding to DNA. A typical incubation condition is 20-30 minutes at room temperature. Since IRDye 700 infrared dye is sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put tubes into a drawer or cover the tube rack with aluminum foil).

  5. Electrophoresis

    • Add 1 μL of 10X Orange loading dye (LI-COR, P/N 927-10100), mix, and load on a gel.
    • Run 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).

  6. Imaging

    Gels can be imaged either inside the glass plates or removed from the plates. 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.

    One of the benefits of Odyssey EMSA analysis is image quantification. However, there are several issues to consider when using the Odyssey Imager to quantify EMSA results.

    IRDye 700 NFkB oligonucleotides were separated on a native polyacrylamide gel

    Figure 1. IRDye 700 NFκB oligonucleotides were separated on a native polyacrylamide gel (4-12% TBE, Invitrogen EC62352BOX) and imaged on the Odyssey Infrared Imaging System.

    • Lane 1) no nuclear extract;
    • Lanes 2) and 5) 10 μg Raji nuclear extract;
    • Lanes 3) and 6) 5 μg Raji nuclear extract

    EMSA sample

    Figure 2. The uppermost shifted band in Lanes 2-7 of Figure 1 was analyzed to determine the level of NFκB binding to the IRDye 700 NFκB oligonucleotides.

    • Free DNA fragments have much lower signal than bound DNA, making quantification of the unbound DNA inaccurate. Addition of 5 mM DTT and 0.5% Tween® 20 to the binding reaction stabilizes the dye and reduces this phenomenon. More Information.
    • In quantification, fluorescent signal of the free DNA band in the control (which contains DNA only and no extract) does not necessarily equal the sum of the signals of the free and bound DNA in samples where extract was added and protein: DNA binding has occurred. This is due to non-specific binding of DNA to components in the nuclear extract.
    • Free oligonucleotides typically form a smear rather than a tight band. This makes it more difficult to precisely quantify bands.

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