IPG strip,Denaturing slab gel,Denaturing See reference: Chinese J ourna1 of Zoonoses (2007) 23(12):1172-1175 See URL: http://www.cqvip.com

Western blotting was performed according to the method of Towbin et al. Proteins were transferred to PVDF membranes using TE77 ECL semi-dry transfer unit (0.8 mA/cm2, 1h). After completion of transfer, non-specific binding sites on the membranes were blocked for 90 min with 5% skimmed milk in TBS at 37℃. Then, PVDF membranes were incubated with primary antibody, rabbit anti-B. melitensis M5 (the agglutination titer of the pooled sera was 1:3200), diluted 1:50 in TBS containing 5% skimmed milk for 1 h at 37℃ on a gentle shaker. The membranes were rinsed in 0.1% Tween20 TBS three times, 10 min each, and incubated with goat anti-rabbit-HRP at a dilution of 1:10000 in TBS containing 5% skimmed milk for 1 h at 37℃. After washing, the blots were developed using ECL Western blotting detection reagents. The specific immunogenic protein pattern was visualized on an X-ray film. [Protocol text in here] [Protocol text in here]