1. Double-strand DNA probe preparation
- Mix the single-strand sense and antisense oligos (100 µM) (ratio 1:1) in a micro-tube (see note 1).
- Place the micro-tube in a heat-block at 95oC for 5 min.
- Afterward, turn off the heat-block. Remove the mental rack containing the micro-tube and let all cool down at room-temperature (see note 2).
- Aliquot and add molecular biology-grade water to dilute the labeled DNA probes to 50 nM (1x) and unlabeled DNA probes to 2500 nM (50x), 5000 nM (100x), 10000 nM (200x), and 15000 nM (300x).
- Store at -20oC for further experiments (see note 3).
2. Native polyacrylamide gel (5%) preparation
- Prepare 5% native polyacrylamide gel as follows:
- Before using, pre-run the gels in 0.5x TBE buffer for 60 min (stable current and 120 V).
3. EMSA reaction
- Each EMSA assay requires at least 7 reactions as illustrated in Table 1.
- Freshly prepare the EMSA mixture as indicated in Table 2 (for one reaction) (see note 4).
- Next, add 1 µl of the unlabeled DNA probes (as shown in Table 3), gently mix up and let the reaction at room-temperature for 15 min.
- Finally, add 1 µl of the labeled DNA probes (50 nM) to each reaction (#1 - #7), gently mix up and let the reaction at room-temperature for 60 min.
- Add 3.5 µl of 6x DNA Gel Loading Dye to each reaction, gently mix up and load an equal amount of each reaction (see note 5) in the gel which was pre-run above.
- Electrophoresis conditions: stable current and 120 V at room-temperature in 0.5x TBE buffer.
- Running time: around 30 min (can stop running when you see the dye reaches 2/3 down length of the gel).
- During electrophoresis, cut the membrane to fit the gel.
- Transferring conditions: stable current and 100 V at room-temperature in 0.5x TBE buffer.
- Transferring time: 30 min.
- After transferring, carefully place the membrane on a holder (e.g. aluminum foil). The gel-contacted surface should be faced up.
- Cross-linking can be carried out in a UV-cross-linker with 254 nm bulbs (120mJ/cm2 for 1 min).
- Subsequently, the membrane can be used for detection or stored at room-temperature for a few days.
- The Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific) can be applied for detection (see note 6).