Semi dry blotting unit

Western blots were utilized to evaluate the sensitivity and specificity of each antibody. Purified proteins (LexA, Eno3, Sdp_SH3b6, Sdp_SH3b2), NV, and total protein extract (TE) collected from L. johnsonii N6.2 under anaerobic and aerobic conditions were loaded at different concentrations (100 ng, 1 µg, 10 µg, and 100 µg). Proteins were separated by SDS-PAGE (10%), at 200 V for 40 mins and transferred at 500 mA for 60 mins onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using a semi-dry blotting unit (Fisher Scientific, Hampton, NH, USA). Membranes were blocked with 5% non-fat dry milk overnight at 4°C. Membranes were washed three times with 0.1% Tween-20 (Fisher Scientific, Hampton, NH, USA) Tris buffered saline (TBS-T). Primary antibodies (anti-LexA, anti-Eno3, anti-Sdp_SH3b2 and anti-Sdp_SH3b6) were incubated in 5% non-fat dry milk overnight at 4°C, at a dilution of 1:1,000. Membranes were washed three times with TBS-T, and the secondary antibody, goat anti-rabbit IgG (H+L) antibody conjugated to horseradish peroxidase (Abcam, Cambridge, MA, USA), was incubated in 5% non-fat dry milk for 1 h at room temperature, at a dilution of 1:2,500. Membranes were washed three times with TBS-T and visualized with ProSignal Femto Solution (Genesee Scientific, San Diego, CA, USA) using the automatic imager FluorChem R (Protein Simple, San Jose, CA, USA).

Samples analyzed with SDS-PAGE (12.5%) were transferred onto a 0.45 μm of polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, United States) using a semi-dry blotting unit (Fisher Scientific, Hampton, NH, United States) at 450 mA for 40 min. The membranes were treated as described before (Loto et al., 2017 (link)). Primary antibodies used were rabbit anti-LotP, rabbit anti-FLAG (1:5,000, Sigma-Aldrich Corp., St. Louis, MO, United States) and mouse anti-His (1:3,000, Sigma-Aldrich). The secondary antibodies conjugated to horseradish peroxidase for chemiluminescence detection were anti-rabbit (1:20,000, Sigma-Aldrich) and anti-mouse (1:10,000, Sigma-Aldrich). Horseradish peroxidase (HRP) activity was detected using Amersham ECL^TM Western Blotting Detection Reagents (GE Healthcare, Pittsburgh, PA, United States). The bands in the membrane were visualized using the automatic imager FluorChem R (ProteinSimple, San Jose, CA, United States), and the band intensity was quantified by ImageJ software (Schneider et al., 2012 (link)).