Agarose Gel Shift Assay for eIF3 Binding

Recombinant eIF3 was expressed and purified from E. coli and native human eIF3 was purified from HeLa cells as previously described^{32 (link)}. The gel shift protocol was adapted from ^{33 (link)} and ^{34 (link)}. A 0.7% agarose gel was prepared using Agarose Type 1B (Sigma A0576) in buffer consisting of 1× TBE supplemented with 75 mM KCl, and gel and buffer were pre-cooled at 4 °C. For each gel shift, 2 μl water, 1 μl of 5× Binding Buffer (25 mM Tris-HCl pH 7.5, 5 mM Mg(OAc)₂, 70 mM KCl, 0.1 mM CaCl₂, 0.1 mg ml^-1 BSA, 2 mM TCEP), 1 μl labeled RNA, and 1 μl of purified eIF3 or protein buffer was added, in the listed order, and incubated at 25 °C for 30 min. 1 μl of room temperature 6× non-denaturing loading dye (40% w/v sucrose, with xylene cyanol and bromophenol blue) was added to the reactions and these were loaded on the agarose gel. The gel was run for 1 h at 40 V at 4 °C, buffer was replaced with fresh cold buffer, and the gel was run for another hour at 40 V. The gel was placed on top of positively charged nylon membrane with four pieces of Whatman filter paper underneath, covered in saran wrap, and dried for 1 h at 75 °C on a pre-heated gel drier. The gel was imaged using a phosphoimager.