Protein Extraction and Enzymatic Assays

One g of the frozen powder (see above) was resuspended at 4°C in 3 mL of 100 mM HEPES (pH 7.5), 2 mM EDTA and 2 mM dithiothreitol, 1 mM PMSF and 10 mL/L protease inhibitor cocktail (Sigma P9599), and centrifuged at 14,000 x g for 20 min. The supernatant was desalted by ultrafiltration on Vivaspin 500 centrifugal concentrator (Sartorius) and the protein extract thus obtained was assayed for enzymatic activities. AGP activity was measured following the two-step assay method described in [57 (link)]. SS activity was measured according to [32 (link)]. One unit (U) is defined as the amount of enzyme that catalyzes the production of 1 μmol of product per min.