Western Blotting Protocol for LtrA Analysis

For LtrA analysis, Western blotting was carried out as described [16 (link), 26 (link)] with modifications. Total cell lysates from low-throughput RTP assay cell pellets were run on a pre-cast 12% polyacrylamide mini-PROTEAN TGX gel (BioRad) at 300 V for 30 min and transferred onto PVDF membrane utilizing BioRad’s Trans-Blot Turbo Transfer System. The membrane was blocked with 10% dry milk, washed, and then incubated with anti-LtrA primary antibody (Covance) at 25 °C for 1 h. The membrane was then washed, incubated with secondary antibody (HRP conjugated, anti-Rabbit (Advansta)) at 25 °C for 1 h, washed again, and protein was detected using Advansta WesternBright Quantum reagents. Membranes were imaged and analyzed on BioRad Chemi Doc XR System. All samples were normalized to total protein per lane on a Coomassie-stained pre-cast gel.