Electrophoretic Mobility Shift Assay (EMSA) for NF-κB and AP-1

Tissue nuclear extract was obtained by using NE-PER nuclear and cytoplasmic extraction reagents (CER I, CER II and NER, Pierce). Tissue was added 150 μl of CER I containing protease inhibitors and homogenized. Sample was vortexed vigorously for 15 sec to fully resuspend the tissue and incubated on ice for 10 min. The mixture was added 11 μl of CER II, vortexed for 5 sec, and incubated on ice for 1 min, then vortexed for 5 sec, and finally centrifuged at 9,500 × g, 4°C for 7 min. The supernatants (cytoplasmic extract) were stored and the pellet containing nuclei was resuspended in 50 μl of NER. The suspension was centrifuged at 9,500 × g, 4°C for 12 min and the supernatant fraction (nuclear extract) was immediately transferred to a clean tube and stored at -80°C until use.

The double-stranded DNA probe containing the NF-κB binding consensus sequence (5’CGCTTCATGACTTGGCCGGAACGCTTG-ATGACTTGGCCGGAA3’) or AP-1 binding consensus sequence (5’CGCTTCATGACTTGGCCGGAACGCTTG-ATGACTTGGCCGGAA3’) were end-labeled with 5 μM biotin-11-UTP (Pierce) at 37°C for 30 min by using terminal deoxynucleotidyl transferase (2 U/μl) and the labeling was stopped by adding 2.5 μl of 0.2 M EDTA.

The biotin-labeled probe was incubated with 5 μg of tissue nuclear protein for binding at room temperature for 20 min. The reaction mixtures were electrophoresed at 100 V on a 4% nondenaturing polyacrylamide gel. The samples were transferred onto the nylon membrane at 380 mA for 1 hr. The membrane was placed to face down on a transilluminator which equipped with 312 nm bulbs and be crosslinked for 15 min. The membrane was washed few times and removed to a new container. The substrate equilibration buffer was added for 5 min with gentle shaking. The cemiluminescent substrate working solution was added and the membrane was placed in a film cassette and exposed to X-ray film for detection.