Nupage novex 4 12 bis tris midi protein gels

Protein extraction was carried out on ice using RIPA buffer (25 mM Tris-HCl pH 7.2, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EDTA, 20 mM NaF, 100 µM orthovanadate), with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, #78430). Proteins were quantified with BCA protein assay kit (Thermo Fisher Scientific, #23225). Total of 50 µg of protein lysates were loaded in each lane of NuPAGE Novex 4–12% bis-tris midi protein gels (Thermo Fisher Scientific, #WG1402A) and proteins were transferred onto nitrocellulose membrane using the iBlot^® 2 Dry Blotting System (Thermo Fisher Scientific). Membranes were cut, blocked in TBS-Tween + 5% milk, and then incubated overnight at 4 °C with the primary antibodies as listed in Table S1. After washes in TBS-Tween, membranes were incubated with the respective secondary antibodies: HRP-conjugated, anti-mouse (#7076S, Cell Signaling Technology, Danvers, MA, USA), or anti-rabbit (#7074S, Cell Signaling Technology). Membrane chemiluminescence was developed with Pierce West Dura substrate (Thermo Fisher Scientific, #34080) and acquired using GBOX (Syngene, Cambridge, UK). Protein band quantification was performed with ImageJ and normalized on the GAPDH signal used as loading control.

Equal amounts of total protein (15 μg per lane) in 1× NuPAGE LDS sample buffer (Thermo Fisher Scientific, NP0007) and 1× Sample Reducing Agent (Thermo Fisher Scientific, NP0009) were loaded per gel lane. Protein samples were electrophoresed on NuPAGE Novex 4–12% Bis-Tris Midi protein gels (Thermo Fisher Scientific, WT1403A) in 1X NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, NP0001–02) at 200 V for 1 h at RT. Gels were transferred to nitrocellulose membranes with an iBlot gel transfer device (Thermo Fisher Scientific). Membranes were blocked with Odyssey blocking buffer (LI-COR, 927–40000) for 1 h at RT, incubated with primary antibodies overnight at 4 °C, washed with TBS containing 0.05% Tween20 (TBST) four times for 5 min at RT, incubated with matching secondary antibodies conjugated to IRDye (LI-COR, 0.1 μg/mL) for 1 h at RT, and washed in TBST four times for 5 min at RT. Protein bands were visualized with an Odyssey CLx Infrared Imaging System (LI-COR) and quantified with Image Studio software (LI-COR).