Anti flag tag rabbit monoclonal antibody

For in vitro secretion, ORFs of XC2081 (avrBs1), XC3176, and XC3002 (hpa1) were cloned into a host-broad plasmid pJXG containing a 3×FLAG-encoding sequence [18 (link)], respectively. Bacteria of Xcc were cultured in rich NYG medium overnight and were transferred into hrp-inducing medium MMX at same cell density for 8 h. The total cell extracts and cultural supernatants were isolated as described previously [31 (link)]. Equal amounts of total cell extract and cultural supernatants were separated by SDS-PAGE and analyzed by immunoblotting, using anti-Flag-tag rabbit monoclonal antibody (1/1000, Sigma). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (1/1000, Pierce) was used as secondary antibody. To ensure that no bacterial lysis had occurred, the protein blots were routinely probed with a rabbit monoclonal antibody specific for β subunit of E. coli RNA polymerase (RNPβ, 1/1000, Abcam, ab12087). Reactions were visualized by enhanced chemiluminescence. All experiments were repeated at least twice.

For Flag‐tagging of NlpD, two fragments containing 798 bp upstream and 730 bp downstream, respectively, of the nlpD termination codon were amplified and EcoRI, BamHI, XbaI and HindIII restriction sites were introduced. The amplified fragments and 3 × Flag oligonucleotides (custom made by Invitrogen) with BamHI and XbaI restriction sites were fused together and cloned into the EcoRI and HindIII restriction sites of the suicide plasmid pK18mobsacB. The recombinant plasmid obtained was introduced into the Xcc wild‐type 8004 by triparental conjugation and the sequence of the wild‐type nlpD gene on the chromosome was replaced with nlpD‐3 × Flag. The resulting strain was named 8004_NlpD‐Flag and was used to investigate the subcellular localization of NlpD (NlpD‐Flag) by Western blot analysis. For this purpose, protein samples were separated in 12% sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel and transferred onto PVDF (polyvinylidene difluoride) membrane (Millipore, Billerica, Massachusetts, USA). After blocking, the 1 : 2000 diluted anti‐Flag‐tag rabbit monoclonal antibody (Sigma) was used as the primary antibody, and the 1 : 5000 diluted horseradish peroxidase‐conjugated goat anti‐rabbit immunoglobulin G (IgG) (Pierce, Rockford, Illinois, USA) was used as secondary antibody.