Mini protean polyacrylamide gel

One aliquot of the eluate was reduced (37 °C, 1 h) and heat denatured (95 °C, 5 min) after addition of an equal volume of 2× sample buffer (150 mM Tris-HCl, 2% SDS, 30% glycerol, 0.04% bromophenol blue, 20 mM Tris(2-carboxyethyl)phosphine (TCEP)). The samples were separated by SDS–PAGE using 4–15% Mini-PROTEAN polyacrylamide gels (Bio-Rad) and 1× Laemmli buffer at constant voltage (100 V) for 75–90 min. The proteins were transferred onto a 0.2-µm PVDF membrane using semi-dry transfer (Trans-Blot Turbo Transfer System, Bio-Rad) and 1× Trans-Blot transfer buffer at 1.3 A and 25 V for 10 min. The membrane was blocked with 5% non-fat milk prepared in PBS containing 0.1% Tween (blocking buffer). Mouse monoclonal IgG against NPM1 (sc-32256, Santa Cruz Biotechnology, clone FC-8791) was diluted in blocking buffer (1:2,000 dilution) and incubated with the membrane overnight at 4 °C. The membrane was washed three times and incubated with goat anti-mouse IgG–HRP (sc-2005, Santa Cruz Biotechnology, 1:5,000 dilution) for 1 h at room temperature. The membranes were washed and developed using an enhanced chemiluminescence kit (Bio-Rad) using the manufacturer’s instructions. Two bands (one from native HeLa cell NPM1 at ~45 kDa and one from heterologous expression of SNAP–NPM1 at ~65 kDa) were detected and quantified using ImageJ (version 1.53e).