Affinity Purification of eIF4F Complex

Cell lysates were prepared from U2OS cells (ATCC, USA) by lysis with NP-50 Lysis buffer (10 mM Tris (pH 8.0), 100 mM NaCl, 0.5% NP-40) and eIF4F was assembled onto m⁷GTP agarose (Jena Bioscience) by tumbling for 2 h. Assembled complexes were further purified by washing with NP-40 lysis buffer to remove unbound proteins^{25 (link)}. Assembled eIF4F complexes were challenged with 100 pmol of indicated RNA for 2 h at 4 °C while tumbling. Displaced proteins were washed away with NP-40 Lysis buffer and bound proteins were eluted with 60 μL of 1X SDS Loading dye. eIF4F displacement was assessed by analyzing eIF4G and eIF4E by western blotting.