One-ml fractions were collected and analysed by SDS–PAGE. The fractions that contained the desired proteins at the expected size were combined and desalted using PD-10 columns (Amersham-Pharmacia) equilibrated in PBS. Purification of recombinant Rv3619c protein. The induction of expression and preparation of cell-free
extract from E. coli BL-21 cells carrying the plasmid pGES-TH/Rv3619c was carried out as described above for Rv3874 and Rv3875. The GST-Rv3619c fusion protein was recovered in the inclusion bodies as pellet, and therefore it was solubilized in successively higher concentrations Ulixertinib supplier of urea in phosphate-buffered saline (PBS), as described previously [20, 25]. Most of the fusion protein was recovered www.selleckchem.com/products/Rapamycin.html in 4 m urea and was purified by affinity chromatography on glutathione-Sepharose column after proteolytic digestion of the column-bound
fusion protein with thrombin protease, as described previously [16, 25]. The fractions that contained the desired protein at the expected size were combined and analysed for purity by 15% SDS–PAGE gels, as described previously . Raising polyclonal antibodies against recombinant proteins in rabbits. Polyclonal antibodies were raised in rabbits against the purified and GST-free Rv3874, Rv3875 and Rv3619c recombinant proteins according to standard procedures . In brief, purified proteins (50 μg/ml) were emulsified with an equal volume of incomplete Freund’s PRKACG adjuvant (Sigma) and injected intramuscularly in the right and left thigh. The rabbits were boosted twice with the same amount of protein at 2 weeks intervals. The animals were bled from the ear before the immunization and 2 weeks after the last immunization. The sera were tested for antigen-specific antibodies using 15% SDS–PAGE gels, as described previously . Enzyme-linked immunosorbent assay (ELISA). ELISA was performed to detect antibodies in rabbit
sera against full-length purified recombinant proteins and overlapping synthetic peptides corresponding to each protein using standard procedures . In brief, wells of 96-well PolySorb plates (Nunc, Rochester, NY, USA) were coated with antigens/peptides (10 μg/ml), blocked with the blocking buffer, incubated with the primary antibody (rabbit sera at 1:100) followed by secondary antibody (alkaline phosphatase-conjugated anti-rabbit immunoglobulin G) and addition of substrate for colour development, as described previously . The colour intensity was measured by determining the optical density (OD) at 405 nm. Antigen-/peptide-coated wells in the presence of secondary antibody alone, i.e. without adding primary antibody, were used as negative controls. The results were expressed as E/C, which is defined as: E/C = OD in antigen-coated wells having primary and secondary antibodies/OD in antigen-/peptide-coated wells having secondary antibody alone. The values of E/C > 2 were considered positive. PCR using gene-specific primers and genomic DNA from M.