No proteases activity detected in E. coli K1 conditioned medium at different pH. Cell-free E. coli K1 conditioned medium was prepared as described in Figure 1A. After electrophoresis, gels were washed for four times with 2.5% Triton X-100 solution for 30 min and incubated overnight in 0.1 M citrate buffer (pH 3, 4 and 5) or 0.1 M phosphate buffer (pH 6, 7 and 8) or 0.2 M glycine buffer (pH 9 and 10). All buffers were added with 100 mM NaCl, 5 mM CaCl2, 500 μM MgCl2 and 2 μM ZnCl2. Following this incubation, gels were stained with Coomassie Brilliant Blue and representative zymographs were visualized. Note that E. coli K1 conditioned medium does not exhibit protease activity at any pH ranging from 3–10. The conditioned medium of Acanthamoeba castellanii was used as a positive control.