Mini protean casting gels

Samples were run on homemade 6–14% Bis-Tris acrylamide gels. Two tubes of 6 ml mix were made up of 6% and 14% acrylamide, respectively (0.33 M pH 6.5 Bis-Tris, 0.083% APS and 0.083% TEMED). Then, 600 μl of the 14% solution was added to 2 × 1 mm Mini-Protean casting gels (Bio-Rad). The 14% solution was diluted with 600 μl of the 6% solution, and a further 600 μl was added to the casting gels. This was repeated until the gels were complete and a comb was added. After polymerization, extracts were loaded to a total of 25–50 µg protein per lane and run at 120 V for 2.5 h at 4 °C. Gels were then transferred onto 0.2 μm nitrocellulose membrane (Bio-Rad, 1620112) using a TransBlot Turbo (Bio-Rad) (30 min, 2.5 Amp). Membranes were stained with Ponceau S solution (Santa Cruz, sc-301558), imaged, cut, washed in TBS and blotted for at least 1 h with TBS containing 5% milk, washed three times in TBS-Tween and incubated with primary antibody overnight. Primary antibody was removed, and membranes were washed three times in TBS-T, incubated with secondary antibody for 1 h, washed three times in TBS-T Tween and imaged on a Chemidoc Touch imaging system (Bio-Rad) using Clarity ECL (Bio-Rad, 170-5061). Where indicated, blots were quantified by densitometry using FIJI with expression of the protein of interest normalized to Ponceau staining (loading control).