EPEC Protein Detection by Western Blot

Sodium Dodecyl Sulfate–Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed as reported previously [58 (link)]. Preparation of EPEC protein extracts are described in the S1 Text. For detection of EPEC proteins by Western blotting, membranes were incubated with primary rabbit antibodies anti-EspB (1:2000), anti-EscC (1:1000), anti-EscJ (1:5000), anti-EscD (1:1000) and anti-Intimin280 (1:5000). Use of polyclonal rabbit sera against EPEC Intimin-280, EscC and EscD were described previously [57 (link), 59 (link)]. Rabbit polyclonal serum against EscJ and EspB was a kind gift of Dr. Bertha González-Pedrajo (UNAM, Mexico). Bound rabbit antibodies were detected with secondary Protein A-peroxidase (POD) conjugate (Life Technologies, 1:5000). GroEL was detected with mAb anti-GroEL-POD conjugate (1:5000; Sigma). Membranes were developed by chemiluminiscence using the Clarity Western ECL Substrate kit (Bio-Rad). The membranes were then developed by exposure to X-ray films (Agfa) or with a Fuji LAS 3000 image when the signal was quantified.