Mini protean tetra cell blotting module

Rhodobacter capsulatus supernatants were concentrated 10-fold using a SpeedVac (Thermo Scientific). Fifteen microliter samples were mixed with 5 μL LDS sample buffer (Abcam). heated to 90°C for 10 min and then run on 4–20% TruPAGE denaturing gradient gel (Merck Life Science Ltd). Proteins were transferred to a PVDF membrane using a Mini-PROTEAN Tetra Cell blotting module (Bio-Rad Laboratories) in 1X transfer buffer (25 mM tris base, 0.2 M glycine, 20% methanol; pH8.5), 100 V for 1 h. The membrane was blocked in 5% (w/v) skimmed milk powder in 1X TBS for 1 h at room temperature. The anti-RcGTA major capsid protein antibody (Agrisera Ltd) was used at 1:1000 dilution in blocking buffer overnight at 4°C, followed by four 10 min washes in TBST. The secondary HRP-antibody conjugate was used at 1:2500 dilution in blocking buffer for 2 h at room temperature, followed by four 10 min washes in TBST. SuperSignal west femto maximum sensitivity substrate (Thermo Scientific) was used to develop the western and the signal was detected using an iBRIGHT chemi-imager (Thermo Scientific).

Western Blot analyses were performed to validate expression changes of the identified LuxS protein detected on the three strains 2D-GE in response to both arsenic conditions. Samples were prepared as described previously and 50 μg of protein supernatant were loaded for SDS-PAGE, followed by its blotting onto a nitrocellulose membrane (Mini-PROTEAN Tetra Cell/Blotting Module – Bio-Rad). LuxS protein was probed primary with a rabbit polyclonal anti-LuxS antibody (1:5000 dilution; MyBioSource: MBS1491336) and a mouse anti-rabbit AP-conjugated was used as secondary antibody (1:5000 dilution; Abcam: 99696). Membranes were digitalized and analyzed with ImageJ v1.52a software (Schneider et al., 2012 (link)) using the digital densitometry values to calculate the expression as fold change.