Standard set

To study the oligomerization state of A1, size exclusion chromatography (SEC) was used by loading 10 μg of purified A1 onto a Superdex 200 (GE Healthcare) size exclusion column. The running buffer contained 50 mM HEPES pH 8.0, 200 mM NaCl and 1 mM DTT. A Bio-Rad standard set was used to generate a standard curve from which molecular weight and oligomerization states of the enzymes were determined. Protein fractions eluted from SEC were resolved on 12% sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) and detected using anti-A1 (1:500, Millipore Sigma) and IRdye labeled goat anti-rabbit antibody compatible with the LI-COR/Odyssey system. The LI-COR/Odyssey software was used to quantitate the relative intensities of A1 in each lane.

The oligomerization states of the enzymes were determined by loading 10 μg of purified enzyme on a 10 mL Superdex 200 (GE Healthcare) size exclusion column. The column was prepared by pouring the resin bed in a column with 16 cm height and 0.5 cm diameter. The running buffer contained 50 mM Tris pH 8.0, 200 mM NaCl and 1 mM DTT. The Bio-Rad standard set was used to generate a standard curve from which molecular weight and oligomerization states of the enzymes were determined.