Mouse monoclonal anti v5 epitope antibody

TCA-precipitated supernatant fractions and pellets were analysed by SDS-PAGE and immunoblotting as described in Kebbi-Beghdadi et al. [36 (link)]. Proteins of interest were detected using a mouse monoclonal anti-V5 epitope antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) (1/5000 dilution) or anti β lactamase antibody (VWR International GmbH, Dietikon, Switzerland) (1/1000 dilution) and MreB was detected using a home-made rabbit polyclonal antibody (1/5000 dilution). First antibodies were incubated overnight at 4 °C. Secondary antibodies, goat anti-mouse IgG-HRP (BioRad, Cressier, Switzerland) and donkey anti-rabbit IgG-HRP (Promega, Dübendorf, Switzerland), both diluted 1/3000 were applied during 1 h at room temperature. Immunoblots were revealed with ECL^TM Prime Western Blotting Detection Reagent (Amersham, GE Healthcare, Glattbrugg, Switzerland) and analysed on ImageQuant LAS4000 mini (Amersham, GE Healthcare, Glattbrugg, Switzerland).

E. coli samples from cultures expressing Waddlia TA modules were collected by centrifugation, and pellets were re-suspended in SDS-PAGE loading buffer. Samples were normalized for equivalent loading using OD_{600 nm} and heated at 95°C for 5 min before loading on SDS-PAGE and transfer onto nitrocellulose membrane (Amersham, Cytiva, Marlborough, MA, USA). Membranes were blocked for 3 h in Tris-buffered saline (10-mM Tris-HCl, 150-mM NaCl) containing 0.05% Tween-20 and 5% dry milk. Proteins of interest were detected using polyclonal mouse antibodies against Waddlia HigB1 or MazF (1/500 dilution), or a mouse monoclonal anti-V5 epitope antibody (Invitrogen, Thermo Fisher Scientific) (1/5,000 dilution) and incubated at 4°C overnight. Horseradish peroxidase-conjugated secondary antibodies, goat anti-mouse IgG (Bio-Rad), and donkey anti-rabbit IgG (Promega) were applied for 1 h at room temperature. Immunoblots were revealed with ECL Prime Western Blotting Detection Reagent (Amersham, Cytiva) using an ImageQuant LAS4000 mini system (Amersham, Cytiva).

Pellet and precipitated supernatant fractions were loaded on SDS gels and proteins were separated by electrophoresis. Immunoblots were performed as described in Kebbi Beghdadi et al.⁵⁸ . Mouse monoclonal anti-V5 epitope antibody (Invitrogen, Thermo Fisher Scientific, Waltham, USA) was used at 1/5000 dilution and incubated for 2 hours at room temperature or overnight at 4 °C. Rabbit polyclonal anti-MreB was used at 1/5000 dilution and incubated overnight at 4 °C. Secondary antibodies, goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP (BioRad, Cressier, Switzerland) were used at 1/3000 dilution and incubated for 1 hour at room temperature. Immunoblots were revealed with ECL^TM Prime Western Blotting Detection Reagent (Amersham, GE Healthcare, Glattbrugg, Switzerland) and analysed on a ImageQuant LAS4000 mini (Amersham, GE Healthcare, Glattbrugg, Switzerland). Image J software was used to quantify signal intensity.