Monoclonal anti flag m2 peroxidase hrp

Proteins were usually separated by standard SDS-PAGE (12% acrylamide) using the minigel system (Bio-Rad). Proteins were denatured by 5 min boiling in Laemmli-buffer containing 70 mM SDS and 2.4 mM 2-mercaptoethanol. The separated proteins were either stained with Coomassie-brilliant blue or transferred onto PVDF membranes (Hybond, Amersham) using electro-blotting for subsequent protein detection with specific antibodies.
Soluble protein extracts (10 μg) from cells of WT, ΔacnSP, or pVZ322_AcnSP, which were cultivated at 100 μmol photons m^–2 s^–1 of continuous light, were used for Western-blotting. For the detection of aconitase a peptide antibody was generated. The synthetic peptide (Ac)-CELLKNPPEAKEEL-amidated specific for AcnB of Synechocystis 6803 was used to produce antiserum in rabbits (Agrisera AB, Sweden). The FLAG-tagged AcnSP protein version in strain pVZ322_AcnSP was detected with a commercial anti FLAG-tag antibody [Monoclonal ANTI-FLAG M2-Peroxidase (HRP), Sigma-Aldrich, United States].

S. aureus cultures harbouring the respective pCN47 or pCN51 derivative plasmids were diluted 1:50 from overnight cultures and grown in TSB at 37°C and 120 rpm until sample collection. For pCN51 constructs induced with CdCl₂, cultures were split at early exponential phase (OD₅₄₀~0.15), 5 μM CdCl₂ was added to half the cultures and incubation continued for another 2 h. S. aureus strains carrying pCN47 reporter constructs were grown to early exponential (OD₅₄₀ ~0.15), mid-exponential (OD₅₄₀ ~0.8) or stationary phase (OD₅₄₀ ~2 after overnight culture). A sample amount corresponding to and OD₅₄₀ of 0.5 in 1 ml was pelleted and stored at -20°C. The sample pellets were re-suspended in 100 μl digestion/lysis buffer (50 mM Tris-HCl, 20 mM MgCl₂, 30% (w/v) raffinose) plus 1 μl of lysostaphin (12.5 μg ml^-1) and incubated at 37°C for 1 h. Samples for SDS-PAGE were prepared by adding 4X NuPAGE LDS Sample Buffer (Invitrogen) and proteins denatured at 95°C for 10 min. Samples were separated by SDS-PAGE (NuPAGE 4–12% Bis-Tris Protein Gels, Invitrogen and then transferred to a PVDF transfer membrane (Thermo Scientific, 0.2 μM) following standard procedures [63 ,64 ]. FLAG-tagged proteins were detected using mouse anti-FLAG-HRP antibody (Monoclonal ANTI-FLAG M2-Peroxidase (HRP), Sigma-Aldrich) following the manufacturer’s protocol.