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).