Western blot
Western blot was performed on API samples and ELISA kit standards using the E. coli HCP WB Kit from Cygnus Technologies (Southport, NC). Various amounts of standards and E. coli control antigens from the kit was loaded onto NuPAGE 4-12% Bis-Tris gel (1.0 mm ×15 well, Invitrogen) after mixing with DTT and 4× LDS sample loading buffer. The proteins were then resolved on the gel by running at 200 V for 35 min. Upon the completion of gel electrophoresis, one gel was stained with Imperial stain solution to visualize protein bands, and a duplicate gel was transferred to PVDF membranes using iBlot (Invitrogen, Carlsbad, CA) apparatus. After transfer, the membrane was blotted using the E. coli HCP WB Kit from Cygnus Technologies (Southport, NC) following the kit’s instructions.