5.2. Cleavage of Stx2a

To obtain the cleaved form of Stx2a, the toxin (4 μg) was treated with 50 ng of trypsin (1 mg/mL in 0.1 mM HCl, diluted to 0.05 ng/mL with PBS) in 10 μL PBS pH 7.5 and incubated for 1 h at 37 °C. Then, to inactivate trypsin, 0.7 ng of the trypsin inhibitor phenylmethylsulfonyl fluoride (PMSF, 1 mg/mL dissolved in absolute ethanol and diluted to 0.7 μg/mL with water) was added and incubated for 10 min at 37 °C [26,30].