Anti groel pod conjugate

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.

A volume of 100 μl of intact bacterial cells or total extracts were absorbed into the ELISA plates (Maxisorb; Nunc) at an OD₆₀₀ of 3.0 and 2 μg/μl, in PBS for 2 h. Next, plates were blocked for 1.5 h with PBS containing 3% (w/v) of skimmed milk. Anti-GroEL POD conjugate (Sigma-Aldrich) was added at 1:5000 dilution to the same buffer and incubated for an additional hour. Anti-Cna mAb mouse custom antibodies were used at 10 μg/ml in the same buffer for 1 h. The plates were then washed five times with PBS, and the presence of bound antibodies were developed using O-phenylenediamine (OPD; Sigma), and absorbance was read at 490 nm. The ELISA values reported were from two independent experiments performed in quadruplicates. The Excel Software was used to create the Graphs of the mean and standard deviation values. Total extracts were obtained from the same culture of intact cells resuspended on PBS and briefly sonicated through three pulses of 20 s (Vibra-cell; Sonics & Materials). All incubations were at room temperature.
The statistical significance of the differences in absorbance measures was evaluated using the Student t-test (^∗p < 0.05, ^∗∗p < 0.01).