Enhanced chemiluminescence reagents

Proteins (400–500 μg) were extracted from the hippocampus and diluted in lysis buffer for further immunoprecipitation using the CoIP kit (Pierce) protocol. Twenty microliters of A/G PLUS-Agarose beads were loaded to a column washed with coupling buffer at 90 g for 1 min. Ten micrograms of rabbit mouse ADNP antibody (Bethyl Laboratories, Montgomery, TX, USA) were added to the beads and incubated for 1 h at 24 °C. Cross-linking using 2.5 mM disuccinimidyl suberate was performed by further 1 h incubation. Then, cleared 500 μg of brain lysate were added to the column and incubated (16 h, 4 °C). To detect the eluted antigen, proteins were separated by electrophoresis on 15% acrylamide gel containing 0.1% SDS,^{10 (link)} transferred to nitrocellulose filter (Millipore, Bedford, MA, USA) and immunostained with rabbit polyclonal antibody against mouse eIF4E (1:500), (a kind gift from Professor Orna Elroy-Stein, Tel Aviv University).^{41 (link)} Proteins were visualized using enhanced chemiluminescence reagents and exposure to hyperfilm (Kodak, Petach Tiqwa, Israel). Protein bands on hyperfilm were quantified using photochromatography analysis.