5′ -- TAC AGA ACA TGT CTA AGC ATG CTG GGG ACT -- 3′
3′ -- ATG TCT TGT ACA GAT TCG TAC GAC CCC TGA -- 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.
p53 is a cellular tumor antigen that functions as a tumor suppresor. For this reason, it is a common oligonucleotide used in EMSA to assess protein binding in cancer and related research.
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 p53 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 |
| 100 mM McCl2 | 1 |
| Water | 12 |
| IRDye 700 p53 | 1 |
| HeLa 4 hour Serum Response 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 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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