Electrophoretic Mobility Shift Assay Protocol

Electrophoresis Mobility Shift Assay (EMSA) was carried out with ³²P-labeled DNA in a total volume of 25 µl in 1× EMSA buffer containing 50 mM NaCl, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 100 µg/ml acetylated BSA, and 3% glycerol. Reaction mixtures containing ³²P-labeled DNA (with or without unlabeled competitor DNA) and proteins were pre-incubated on ice (unless otherwise indicated) for 30 min. Super-shift experiments were carried out by addition of 0.5–1 µL of specific antibody to the pre-incubated protein-DNA complexes, followed by 20 min incubation on ice (in the presence of a 1000-fold mass excess of unlabeled competitor DNA over ³²P-labeled DNA). Reaction mixtures were finally loaded on pre-run 5% or 8% polyacrylamide gels (29:1 acrylamide/N,N′-methylenebisacrylamide) in 0.5x TBE containing at 200 V (4°C) for 4–6 h. Following the electrophoresis, the gels were dried, and the DNA visualized and quantified on PhosphorImager Typhoon SLA9000 (GE). The dissociation constant K_d was approximated as the total protein concentration at the point of the titration where the fraction of the protein-bound DNA was 0.5 [9] (link), [19] (link).