Nupage novex 4 12 tris acetate gel

Purified protein eluted from 1 ml HiTrap Heparin HP affinity column (GE) was fixed in NuPAGE LDS sample buffer (Thermo Fisher Scientific) at 98°C for 5 min and complexes were resolved by running 8 μl from each sample on a NuPAGE Novex 4–12% Tris-acetate gel (Thermo Fisher Scientific). Proteins were transferred to a PVDF membrane using Turbo-Blot transfer apparatus and Trans-Blot TurboTM Midi PVDF Transfer Packs (Bio-Rad). The membrane was blocked with PBS + 5% milk for 1 h at room temperature then incubated with a PBS + 1% milk solution containing a 1:2000 monoclonal anti-FLAG antibody (Sigma-Aldrich #F3165) for 1 h at room temperature. The membrane was washed three times with PBS + 0.05% Tween-20 and then incubated with PBS + 1% milk solution containing a 1:10 000 anti-mouse horseradish peroxidase conjugated secondary antibody (Sigma-Aldrich #A9044). The membrane was washed three times with PBS + 0.05% Tween-20 and then incubated for 5 min with Pierce ECL Plus substrate (Thermo Scientific). Chemiluminescence was detected using an ImageQuant LAS 4000 imager (GE Healtcare). Images were processed using Fiji (https://doi.org/10.1038/nmeth.2019).