Endotoxin removal/LAL assay
Endotoxin was removed from all proteins using Endotoxin removal columns (Pierce, Thermoscientific) as per the manufacturer’s protocol. Briefly, 4-6 mg of protein diluted to 2 ml in PBS buffer was incubated in a prepared column at room temperature for 6 hours or at 4°C for 18 hours prior to elution. The procedure was repeated four times consecutively for each protein with the columns regenerated in between. Endotoxin levels were determined by LAL assay (Pierce, Thermoscientific) following the manufacturer’s instructions. Chromogenic readings at OD 405nm were taken using a Clariostar plus plate reader (BMG Labtech).