Immunoblotting of Rv1636 Protein in Mycobacteria

Protein samples were electrophoresed on SDS-polyacrylamide gel of appropriate percentage and transferred to polyvinylidene fluoride membrane for immunoblotting. Blots were probed with the primary antibody in TBST (10 mM Tris–Cl (pH 7.5), 0.9% NaCl, and 0.1% Tween 20) containing 0.2% bovine serum albumin (TBT) overnight at 4 °C followed by horseradish peroxidase (HRP)-conjugated secondary anti-rabbit immunoglobulin G antibody (1:50,000) for 1 h at room temperature (RT). Bound antibody was detected using enhanced chemiluminescence with Immobilon Western HRP Substrate (Merck Millipore). A total of 20 to 25 μg of protein was loaded for whole cell lysates and sub-cellular fractions, whereas 25 ng was loaded for purified recombinant Rv1636. Antisera against Rv1636 was raised by immunizing a rabbit with purified, recombinant Rv1636. Anti-CRP (59 (link)) and anti-CFP-10 (BEI Resources, NIAID, National Institutes of Health, USA) antisera were used at recommended dilutions. Anti-CRP and anti-GyrB antibodies were described earlier and available in the laboratory (59 (link), 60 (link)).