Pull-down of nuclear proteins from HGC-27 cell line

To identify nuclear proteins that interact with KIT2KIT* G-quadruplex DNA, pull-down assays were performed with nuclear extracts from the KIT-positive HGC-27 cell line. Streptavidin-coated paramagnetic beads were derivatized with the biotinylated oligonucleotide and subsequently incubated with nuclear extracts. Bound proteins were eluted with a KCl gradient. A last fraction was obtained by boiling beads in denaturing Laemmli sample loading buffer. When solved by SDS-PAGE, this last fraction exhibited three main bands that were cut and subjected to in-gel trypsin digestion and LC-MSE analyses for protein identification. Two of them (bands S1 and S2 at ~15 kDa and ~28 kDa, respectively) corresponded to Streptavidin monomer and dimer that detached from the beads along boiling procedure. That aside, the band at ~50 kDa (band V) was associated to the intermediate filament protein Vimentin.