Alkaline phosphatase conjugated anti rabbit antibody

Whole cells lysates were prepared by pelleting 1 ml of an overnight culture diluted to an optical density at 600nm (OD₆₀₀) of 1.0, and resuspending in 50 μl of distilled water plus 50 μl of 2x SDS loading buffer. SDS PAGE and transfer of proteins to a PVDF membrane for western blotting was performed as previously described [53 (link)]. Monospecific antiserum against H4 flagellin was purchased from the Statens Serum Institute, Denmark. OmpA antiserum was purchased from the Antibody Research Corporation, USA (item #111120). Primary antibodies were detected with commercially purchased alkaline phosphatase-conjugated anti-rabbit antibody (Sigma Aldrich). SIGMAFAST^™BCIP^®/NBT (Sigma Aldrich) was used as substrate for detection.

Whole cells lysates were prepared by pelleting 1 ml of an overnight culture diluted to an optical density at 600nm (OD₆₀₀) of 1.0, and resuspending in 50 μl of distilled water plus 50 μl of 2x SDS loading buffer. SDS PAGE and transfer of proteins to a PVDF membrane for western blot were performed as previously described71 (link). Monospecific antisera against H1, H4 and H7 flagellin was purchased from the Statens Serum Institute, Denmark. Polyclonal antibodies against FimA (targeting the peptide CAGSVDQTVQLGQVRT) and LafA were generated by the Antibody Facility at the Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia). OmpA antiserum was purchased from the Antibody Research Corporation, USA (item #111120). Primary antibodies were detected with commercially purchased alkaline phosphatase-conjugated anti-rabbit antibody (Sigma Aldrich). SIGMAFAST^TMBCIP®/NBT (Sigma Aldrich) were used as substrate for detection.

Genomic DNA was isolated by CTAB method from PCR positive p68 tobacco transgenic lines and WT. For southern blots analysis, ∼20 µg of genomic DNA was digested with HindIII and resolved on agarose gel. The DNA was transferred to nylon membrane (Hybond N, Amersham Pharmacia, http://www.gelifesciences.com/), and hybridized with radiolabelled p68 cDNA as described previously [12] . For western blot analysis, the total soluble proteins were isolated from the unstressed tissue samples of transgenic lines as well as WT plants and separated on 12% SDS-PAGE. Western blot analysis was performed by using anti-p68 (1∶5,000 dilutions) as primary and anti-rabbit (alkaline phosphatase conjugated antirabbit antibody-Sigma) as secondary antibody (1∶12,500 dilutions). The blot was developed as per manufacturer’s protocol (Sigma, USA).