Lipid overlay blots with SidP, MavQ, and Lem14

6xHisSBP‐tagged SidP, MavQ, and Lem14 were purified using the 6xHis‐tag, as described above, followed by an additional SBP‐tag purification step with streptavidin mag sepharose (GE Healthcare Life Sciences) and eluted in 50 mM HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol, and 4 mM biotin. The lipid overlay blot was performed as described (Weber et al, 2013), with minor modifications. Briefly, the PIP strips (Echelon, P‐6001) were blocked in 3% fatty acid‐free BSA (Roche) in Tris‐buffered saline with 0.1% Tween 20 (blocking buffer) and incubated overnight in the dark at 4°C with 5 pmol/ml purified protein in a total volume of 10 ml blocking buffer. The PIP strips were washed with blocking buffer, incubated with mouse anti‐polyhis antibody (1:2,000, clone HIS1, Sigma‐Aldrich) in blocking buffer, washed, and incubated with anti‐mouse HRP antibody (7076, Cell Signaling Technologies) in blocking buffer.