Fig. 3

Protease inhibitor assay with spent supernatant of B184 and Affibody molecule standards. SDS-PAGE of spent supernatant with the indicated Affibody molecules and addition of different protease inhibitors (cropped). The supernatant is derived from a cultivation of B184k producing α-amylase grown for 24 h at 30 °C at 220 rpm. The Affibody molecule standards were incubated overnight at 30 °C at 220 rpm. The lane marked with pc contains the positive control with spent supernatant with the respective Affibody molecule and the complete protease inhibitor cocktail, the lane marked with nc contains the negative control with spent supernatant with the respective Affibody molecule, the lane marked with st contains the standard of the respective Affibody molecule in water, the lane marked with AEBSF contains spent supernatant with the respective Affibody molecule with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride 1 mM, the lane marked with aprotinin contains spent supernatant with the respective Affibody molecule with aprotinin 6.5 μg/mL, the lane marked with bestatin contains spent supernatant with the respective Affibody molecule with bestatin hydrochloride 50 μM, the lane marked with E-64 contains spent supernatant with the respective Affibody molecule with E-64 15 μM, the lane marked with leupeptin contains spent supernatant with the respective Affibody molecule with leupeptin 20 μM and the lane marked with pepstatin A contains spent supernatant with the respective Affibody molecule with pepstatin A 10 μM