Abstract
Western blotting is a fundamental technique for protein separation and identification based on molecular weight. However, the detection of Antigen 43 (Ag43) in Escherichia coli is limited by non-specific primary antibodies, resulting in streaky blots with multiple bands. Here, we present an optimized protocol for the specific detection of Ag43 expression in E. coli whole-cell lysates transformed with pBAD-Ag43 plasmid encoding Ag43(full) and the pSMAK plasmid encoding Ag43(∆193-551). Following the induction or repression of Ag43 expression using arabinose and glucose, respectively, whole-cell lysates are obtained and subjected to SDS-PAGE, membrane transfer, and an optimized antibody incubation process prior to fluorescence imaging. This method reliably distinguishes unprocessed Ag43(full) at ~103 kDa, processed Ag43(full) at ~55 kDa, and Ag43(∆193-551) at ~40 kDa, while distinguishing these bands from those in cells transformed with control plasmids such as pBAD24. Our approach provides a robust framework for the confirmation of Ag43 expression in transformed E. coli strains, addressing key limitations in specificity and reproducibility.