Amersham protran premium 0.45 nc

Prepped cell lysates were separated by SDS-PAGE using 10% or 12.5% gels. Protein sizes were estimated using the PrecisionPlus All Blue protein marker (Bio-Rad, USA). Gels were incubated in Bjerrum Schafer-Nielsen transfer buffer and proteins were transferred to water-activated nitrocellulose membranes (Amersham Protran Premium 0.45 NC, GE Healthcare Life Sciences, Sunnyvale, USA) using semi-dry transfer (Trans-Blot® Turbo™, Bio- Rad, USA). Next, the membranes were stained in Ponceau solution for 5 min and scanned to be used as a loading control. The membranes were blocked in 5%-10% skim milk in TBS-T (Fluka, Taufkirchen, Germany) for 1 h at RT. After blocking, the membranes were incubated with the respective primary antibodies diluted in 1% Bovine Serum Albumin (BSA) or 5% skim milk overnight at 4 °C with gentle agitation. Next, the membranes were rinsed 3 × 5 min with TBS-T, incubated 1 h at RT with HRP-conjugated secondary antibodies (R&D Systems) followed by rinsing 3 × 5 min with TBS-T. Chemiluminescence was detected using an ECLplus kit (GE Healthcare, Amersham™) and an Azure c500 system (Azure Biosystems, Dublin, USA). Protein band quantification was carried out using ImageJ software (NIH, http://rsb.info.nih.gov/ij/). All antibodies used are listed in Supplementary Table 5.

Purified proteins were analysed by SDS–PAGE [33 (link)]. Coomassie Brilliant Blue was used to stain protein bands. Protein contents in bacterial lysates were determined using the Quant-iT™ Protein Assay Kit and Qubit^® fluorometer (Invitrogen). Proteins were electroblotted onto nitrocellulose blotting membranes (Amersham Protran Premium 0.45 NC, GE Healthcare) for western blot analysis. MabR_MS was detected using 1 : 100 dilution of polyclonal anti-MabR_MS serum and antigenic polypeptides were visualized using a horseradish-peroxidase-conjugated secondary antibody. To identify RpoB_MS, the membrane was probed with a 1 : 20 000 dilution of polyclonal anti-RpoB_TB serum, and antigenic polypeptides were visualized using an alkaline-phosphatase-conjugated secondary antibody. Antiserum against His₆-MabR_MS was elicited in rabbits and anti-RpoB in mice.

Total supernatant was separated on a 12.5% SDS polyacrylamide gel, and blotted onto Amersham Protran Premium 0.45 NC (GE Healthcare Life Sciences). The membrane was blocked overnight at 4°C in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T, Sigma-Aldrich) and 5% non-fat dry milk (AppliChem). After washing with 0.05% TBS-T, the membrane was incubated with a polyclonal rabbit anti-TTR antibody (S2 Table) diluted 1:500 and incubated for two hours at room temperature. Horseradish peroxidase (HRP)-linked secondary antibody was used (GE Healthcare Life Sciences). Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences) was added. Blots were quantified using ImageJ 1.48v analysis software. TTR of cell culture supernatants was analyzed using liquid chromatography-high resolution accurate mass MS (LC-HRAM MS) in positive-ion mode.