Streptavidin-biotin affinity pull-down assay

MDA-MB-231 cells were harvested, and lysates prepared and incubated with free biotin (MedChemExpress, Monmouth Junction, NJ, USA) or MEQ-biotin (10 μM) with rotation for 5 h at 4 °C. Recombinant streptavidin agarose beads were then added and incubated with rotation at 4 °C overnight. The beads were washed three times and bead-bound proteins separated by SDS-PAGE. Protein bands were visualized by silver staining. Bands between 70 kDa and 120 kDa were cut out for mass spectrometric analyses. MTA2 was detected using western blotting of the same streptavidin agarose bead-binding proteins.