Bands corresponding to the DNA/protein complex and free probe were extracted from an EMSA gel and were run in a 20% denaturing acrylamide sequencing gel.
A. The bromine footprint of the free wild-type sequence represented by the HC27 oligonucleotide were it showed a typical i-motif footprint at pH 6.5 in lane I. Lane II corresponds to the HC27 probe complexed to hnRNP K where it shows a footprint distinct from that of an i-motif in lane I. Differences in the bromine footprints are highlighted by four solid arrows showing diminished bromine protection in lane II and one open arrow that demonstrates the bromination of a cytosines unaffected by the DNA/protein complex.
B. The bromine footprint of the free CmidT mutant probe in lane III demonstrating complete bromination of cytosines throughout the oligonucleotide. Lane IV is the EMSA/footprinting non-specific protection pattern of CmidT bound to hnRNP K.
