Anti cfp 10

The bacteria were incubated for 3 weeks in a medium containing a reduced amount of bovine serum albumin (0.05%), and subsequently, the supernatants containing secreted proteins were obtained by centrifuging at 3000 g for 15 min. Secreted proteins were precipitated with 10% trichloroacetic acid overnight at 4°C and centrifuged at 7000 g for 30 min at 4°C. The proteins were resuspended in cracking buffer (60 mM Tris-Cl pH 6.8, 2% sodium dodecyl sulfate [SDS], 10% glycerol, 2% β-mercaptoethanol, and 0.01% bromophenol blue). For Western blot analyses, the samples were subjected to electrophoresis in 15% SDS-polyacrylamide gel electrophoresis gels, then transferred to nitrocellulose membranes, and finally stained with Rouge Ponceau solution. The membranes were blocked with TBS (10 mM tris-HCl pH 7.5 and 150 mM NaCl) supplemented with 5% skim milk for 30 min before incubating them with primary rabbit polyclonal antibodies (anti-ESAT-6 or anti-CFP-10, BEI Resources) in a dilution 1/100 for 2 h. The nitrocellulose membranes were washed with TBS thrice and incubated with a secondary anti-rabbit alkaline phosphatase-conjugated antibody at a 1:10,000 dilution for 2 h. Western blots were revealed by incubation with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium solution.