CTCF gel-shift chemiluminescent EMSA assay (electrophoretic mobility shift assay)

The CTCF gel-shift chemiluminescent EMSA assay was carried out with the Gel-shift Chemiluminescent EMSA Assay Kit (Active Motif, catalog no. 37341). The DNA oligo (purchased from Eurofins Genomics) sequence used was 5’-gtagtgacccCGgagccaCGtggagccaccttcagacccagagctgaagcaggagggtgacCGg-3’. The biotin-dT label was on the 5’ end of the reverse strand. The CpGs that were either un-methylated or fully methylated are in underlined capitals. The CTCF-binding motif is in italic font. The EMSA assay was carried out following manufacture’s instruction with some modifications. The final concentration for the labeled dsDNA was 2 nM. NP-40 was added to the final concentration of 0.02%. 50 ng/µL Poly d(I-C) was used as a non-specific competitor. Maltose-binding protein-fused (MBP-fused) CTCF zinc finger 1–11 protein was over-expressed in E. coli and purified with amylose resin (New England Biolabs, catalog no. E8021S) according to the manufacturer’s protocol. The details of the CTCF preparation procedure were described in a previous publication⁵⁴.