Western Blot Analysis of AcrA in K. pneumoniae

Western blotting was performed as previously described, with some modifications [57 (link)]. Overnight cultures of K. pneumoniae were centrifuged and resuspended in 1 mL 50 mM Tris/HCl (pH 8.0) and ultrasonically lysed on ice (four 30-second pulses, with a 30-second pause between each pulse). The Omni-Easy Instant BCA Protein Assay Kit (EpiZyme) was used to quantify protein concentration. Proteins were electrophoresed in Tris-glycine-sodium dodecyl sulfate electrophoresis buffer (EpiZyme). Separated proteins were transferred to PVDF membranes in Omni-Flash transmembrane buffer (EpiZyme). After washing, the membranes were incubated with antibodies against AcrA (custom-made by Abmart) and GAPDH (GeneTex) overnight (GAPDH was used as an internal reference). The membranes were washed and incubated with an enzyme-linked rabbit secondary antibody (Absin). Protein bands were captured using an enhanced chemiluminescence (ECL, EpiZyme) western blotting detection system (Tanon) and densitometry was performed using Tanon Image software (Tanon).