E. coli K1 did not affect degradation of bovine serum albumin (BSA) and rabbit immunoglobulin G (rIgG) in protease assays. The conditioned medium of E. coli K1 strain E44, E. coli K-12 strain HB101 and A. castellanii was prepared as described in Materials and Methods. The conditioned media were incubated with 2 μg of BSA (A) and rIgG (B) overnight in incubation buffer (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 5 mM CaCl2, 500 μM MgCl2 and 2 μM ZnCl2) at 37°C. Next day, an equal volume of 2× SDS sample loading buffer was added in all samples, boiled and loaded onto 12% SDS-PAGE gel. After electrophoresis gels were stained with Coomassie Brilliant Blue and visualized. Note that bacterial conditioned media did not degrade BSA/rIgG in protease assays tested. The results are representative of three independent experiments.