Native page novex 3 to 16 bis tris gels

A 10× loading dye (5% w/v Coomassie Blue G-250, 500 mM 6-aminohexanoic acid) was added to 20 μg of purified C-terminal MRAP2 domain and loaded on Native PAGE Novex 3% to 16% Bis–Tris gels (Thermo Fisher, Life Technologies, Inc, Milan, Italy). After electrophoresis at 0 °C (using buffers and conditions specified by the manufacturer and reported in [16 (link)]), proteins were transferred to a polyvinylidene fluoride membrane (60 min at 15 V), which was pre-wetted in methanol for 30 s and then soaked in transfer buffer for several minutes [16 (link)]. The membrane was decolorized in methanol for 3 min, rinsed in T-TBS pH 7.4, blocked, and immunoblotted with monoclonal anti-His (1:5000, Invitrogen-Thermo Fisher Scientific, Milan, Italy).

Blue native-PAGE of purified MRAP2-CT, R/H-CT, R/C-CT was performed on NativePAGE Novex 3% to 16% Bis-Tris gels (Life Technologies—Thermo Fisher Scientific, Waltham, MA, USA) as described in Fullone et 2022 [5 (link)] using the buffers and conditions specified by the manufacturer and described in [23 (link)]; 20 µg of each protein sample was analyzed.