EMSA

From Genome@Yale

• Liquid chemiluminescent DNA pull-down assay to measure nuclear receptor-DNA binding in solution

SYBR-EMSA

1. Anneal oligos - do this prior to setting up the EMSA.
2. A serial dilution of the recombinant protein (0.125 μg, 0.25 μg, 0.5 μg and 1.0 μg - the actual dilution series depends on the affinity of the protein to the DNA) is incubated with 1.5 pmoles of oligo in binding buffer (12.5 mM Hepes, pH 8.0, 50 mM NaCl, 5 mM DTT, 5% glycerol, and 1 mg/mL BSA) for 30 minutes at room temperature. Mix well. Too much protein will interfere with UV.
3. The reaction mixture is gently loaded onto 6% (29:1 mono:bis) polyacrylamide gel containing 1X TBE (89 mM Tris, 89 mM Boric Acid, and 2 mM EDTA). This step is critical.
4. The polyacrylamide gel was subjected to electric current of 5 V/cm for 1 hour at room temperature, stained with SYBR green I DNA dye and visualized under UV illumination.