Fig. 1

Schematic overview of the protein lipid overlay (PLO) assay. a Schematic overview of the classical protein lipid overlay (CPLO) assay. Different types or concentrations of lipids are spotted onto a PVDF membrane, blocked with PBST blocking buffer comprising 0.1% Tween 20 (v/v) and BSA (3%, m/v) or 0.1% Tween 20 (v/v) and skimmed milk (5%, m/v), then incubated with epitope-tagged (GST, MBP, His, etc.) proteins of interest at concentrations of 1 − 5 µg/mL. After washing and incubation with primary and secondary antibodies, proteins bound to the membrane are detected by enhanced chemiluminescence (ECL) according to the manufacturer’s instructions. b Schematic overview of the modified protein lipid overlay (MPLO) assay. Different types or concentrations of lipids are spotted onto a PVDF membrane, and the membrane is simultaneously blocked and incubated with epitope-tagged (GST, MBP, His, etc.) proteins of interest in PBST blocking buffer comprising 0.1% Tween 20 (v/v) and BSA (3%, m/v) or 0.1% Tween 20 (v/v) and skimmed milk (5%, m/v). After washing and incubation with primary and secondary antibodies, proteins bound to the membrane are detected by enhanced chemiluminescence (ECL) according to the manufacturer’s instructions. More details are provided in “Materials and methods”