Evaluating BbZIP Protein Expression in E.coli

E.coli cells (C43 strain) transformed with the empty vector or the plasmids harboring the genes encoding BbZIP or the variants were allowed to grow in the autoinduction media overnight before they were applied on LB agar plates containing indicated concentrations of ZnCl₂. After 24 hours of incubation at 37°C, the plates were imaged. To estimate expression of BbZIP and the variants, cell lysates were mixed with SDS sample loading buffer, applied to SDS-PAGE, and transferred to a PVDF membrane. After being blocked with 5% nonfat dry milk, the membrane was incubated with a mouse custom anti-BbZIP monoclonal antibody at 0.06 µg/ml at 4°C overnight (32 (link)). The bound primary antibodies were detected with an HRP-conjugated anti-mouse immunoglobulin-G at 1:5000 dilution (Cell Signaling Technology, Catalog# 7076S) by chemiluminescence (VWR). The images of the blots and plates were taken using a Bio-Rad ChemiDoc Imaging System.