5′ -- AGT TGA GGG GAC TTT CCC AGG C -- 3′
3′ -- TCA ACT CCC CTG AAA GGG TCC G -- 5′
Underlined nucleotides are the binding site. IRDye 700 oligonucleotides are supplied as 25 µL of 50 nM (or 50 fmol/µL) double-stranded DNA.
NFκB is a transcription factor that controls transcription of DNA, cytokine production, and cell survival. For this reason, it is a common oligonucleotide used in EMSA to assess the binding of specific proteins.
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 NFĸB oligonucleotide, the following binding reaction is a good starting point:
| Reaction | µL |
|---|---|
| 10X Binding Buffer (100 mM Tris, 500 mM KCI, 10 mM DTT; pH 7.5) | 2 |
| Poly (dl•dC) 1 µg/µL in 10 mM Tris, 1 mM EDTA; pH 7.5 | 1 |
| 25 mM DTT/2.5% Tween® 20 | 2 |
| 1% NP-40 | 1 |
| Water | 12 |
| IRDye 700 NFĸB | 1 |
| Raji nuclear extract (positive control) (5 µg/µL) in Dilution Buffer (20 mM Hepes (pH 7.9), 100 mM KCI, 1 mM MgCl2, 20% glycerol, 0.5 mM PMSF, and 0.5 mM DTT) | 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 infrared dyes are somewhat sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put the tubes into a drawer or simply cover the rack containing tubes with aluminum foil). After the incubation period, 10X Orange Loading Dye (P/N 927-10100) is added to the binding reaction for electrophoresis.
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