Quantifying mRNA-binding Protein Interactions

Flag antibody (M185-3L, MBL) was diluted with antibody coating buffer [carbonate buffer (pH 9.6)] to 1 µg/ml to cover the 96-well plate at 4°C overnight. The coated plate was washed with PBST (PBS supplied with 1% Tween-20) to remove the unbound antibody. Purified inactive mR3 proteins were firstly diluted with storage buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl] (Glow et al., 2015 (link)), followed by dilution with PBST to reaction concentration (0.8 µM for monomer proteins, 0.4 µM for dimer proteins). The diluted proteins were transferred to an antibody-coated plate to incubate for 2 h at room temperature, followed by washing with PBST. Biotin-labeled dsRNAs were diluted with LSB [10 mM Tris-HCl (pH 7.5), 5 mM NaCl, 1 mM MgCl₂, 0.1 mg/ml bovine serum albumin] or HSB [20 mM HEPES-KOH (pH 7.5), 140 mM KCl, 12 mM NaCl, 2 mM MgCl₂, 5% glycerol] to 0.02 µM to bind with coated inactive mR3 proteins for 1 h at 37°C, followed by washing with PBST and incubating with Streptavidin-HRP antibody (Abcam, ab7403, 1:1000) at 4°C overnight. After incubation and washing, the plate was stained with TMB Chromogen Solution (Beyotime, P0209) at 28.5°C. After sufficient color development, the staining reaction was stopped with 2 M H₂SO₄. The staining result was read out by microplate reader (EnSpire, PerkinElmer) at 450 nm.