High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

Binding assays were carried using either WT Dicer or the catalytically-inactive Dicer variant (D1320A/D1709A). Dicer D1320A/D1709A was first diluted in EMSA buffer (100 mM Tris pH 7.6, 100 mM NaCl and 20% glycerol), whereas WT Dicer was diluted in EMSA buffer supplemented with 1 mM EDTA. In parallel, 32 P-labeled pre-let-7a-1 was refolded by heating at 95°C for 2 min and snap-cooling at 4°C for a minimum of 5 min. The RNA was then diluted in 0.1% NP-40 and 4 mM DTT to a concentration of 4 pM. The binding reactions were initiated by adding 10 μL of the RNA sample to 10 μL of the protein samples. Standard binding reactions contained 50 mM Tris pH 7.6, 50 mM NaCl, 10% glycerol, 0.05% NP-40, 2 mM DTT, and 2 pM 32 P-labeled RNA (0.5 mM EDTA was added for reactions with WT Dicer) with protein concentrations varying between 0.01× and 100× of the estimated K d value. Reactions were incubated for 30 min at 4°C and loaded on a 4-15% gradient polyacrylamide gel (37.5:1 acrylamide:bis-acrylamide) in Tris-Glycine buffer (25 mM Tris-Base and 200 mM glycine), which was run for 2 h at 200 V in the cold room (4°C). Gels were dried, exposed overnight to a storage phosphor screen (Bio-Rad) and visualized with a Personal Molecular Imager (Bio-Rad). Band intensities for the bound (B) and unbound (U) RNA were quantified using the ImageLab software version 5.2 (Bio-Rad). The fraction of bound RNA [F = B/(B + U)] was plotted against protein concentration, and the binding data were fitted to the Hill equation with the OriginPro 8 software (OriginLab). The reported K d value and its errors represent the average and standard deviation from two independent experiments.