Aaf amc

Cells cultured to 70-80% confluent were harvested and washed thrice in PBS. Then, cells were resuspended in 4 volumes of freshly prepared homogenization buffer (50 mM Tris-HCl, pH7.5, 250 mM Sucrose, 5 mM MgCl2, 0.5 mM EDTA, supplemented with 2 mM ATP, 1 mM DTT and 0.025% Digitonin before use) and incubated on ice for 5 min to allow permeabilization by digitonin. Cytosol was squeezed out by centrifugation at 20,000 g for 15 min at 4°C and transferred to a new tube. Protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). In between, 200 μM (2 X) AAF-AMC (MERCK, Darmstadt, Germany) substrate solution with or without 100 μM (2 X) AAF-CMK (MCE, Monmouth Junction, USA) was prepared by diluting 100 X stock solutions (100 mM AAF-AMC in anhydrous ethanol, 50 mM AAF-CMK in ddH2O) in the assay buffer (50 mM Tris-HCl, pH7.5, 40 mM KCl, 5 mM MgCl2, supplemented with 0.5 mM ATP, 1 mM DTT and 0.5 mg/mL BSA before use). Then, the 2 X substrate solution was transferred into a well of 96 well plate and incubated at 37°C for 5 min. At last, cytosol equal to 5 μg proteins was added to the well and assay buffer was supplemented to 200µL total volume. The fluorescent signal was recorded with Infinite 200 PRO (Tecan, Männedorf, Switzerland) at Ex 380/Em 460 nm.