Chemidoc mp imaging system universal hood 3

EMSA was performed with purified recombinant p50 protein and Cy5 labeled oligonucleotide probes. Oligonucleotide probes used in the EMSA were the following: probe C-For: 5’ Cy5-CCCCBrdUGCCCT-CAGTGGGCAGCCTCTGCATTCCCBrdUCAGCTCCCTTTCTCTCTGTGA-3’ OH; Probe C-Rev: 5’ TCACAGAGAGAAAGGGAGCTGAGGGAATGCA-GAGGCTGCCCACTGAGGGCAGGGG-3’ OH; Probe G-For: 5’ Cy5 CCCCBrdUGCCCTCAGTGGGCAGCCTCTCCATTCCCBrdU-CAGCTCCCTTTCTCTCTGTGA-3’OH; Probe G-Rev: 5’-TCACAGAGA-GAAAGGGAGCTGAGGGAATGGAGAGGCTGCCCACTGAGGGCAGGGG-3’ OH; C-oligo NF-κB -For: 5’-CCGCTGGGACTTTCCAGGA-3’ OH; C-oligo NF-κB -Rev: 5’-TCCTGGAAAGTCCCAGCGG-3’ OH. Probes were annealed at 90 ^°C and allowed for slow cooling at room temperature overnight. For each binding reaction 1x binding buffer was used containing 50 mM Tris (pH 8.0), 200 mM KCl, 5 mM EDTA, 5 mM DTT and 0.25 μg/microliter BSA (w/v). A typical binding reaction was set up in 20 μl volume and contained 1 μl of labelled probes (5 uM), 6 μl of recombinant protein and 1 μl of cold competitor (5 uM C-oligo). DNA binding reaction was performed on ice for 30 min. 1 pl of 6x DNA loading dye was added to each reaction and run on 4% native PAGE for 50 min at 4 ^°C. The gel was imaged using Biorad Chemidoc MP imaging system (Universal hood III).