Immunoblotting
Recombinant purified protein (10 µl) or eluates (10 µl) and 2× Laemmli buffer (Sigma) (10 µl) were run on a precast Criterion Any kD TGX Stain-Free gel (BioRad) at 150 V for 1 h. Gels were blotted onto prepackaged Iblot2 stacks (PVDF membrane, Invitrogen) using a custom Iblot2 program (20 V 3 min, 23 V 1 min, 25 V 1 min). Membranes were blocked for 15 min in SuperBlock, TBS (Thermo-Fisher Scientific). Recombinant proteins were detected using mouse anti-6x-His antibody (Clonetech) [1:20,000 1×TBST (1.5 M NaCl, 10 mM Tris-HCl pH 7.5, 0.05% tween 20), 1 h at room temperature] and goat anti-mouse HRP (1:20,000 1×TBST, 1 h at room temperature) (Seracare). PlzA protein was detected using rat anti-PlzA antibody (1:5,000 1×TBST, 1 h at room temperature) and goat anti-rat antibody (BioRad) (1:40,000 1×TBST, 1 h at room temperature). Membranes were washed with 1×TBST three times for 5 min between antibodies and before detection. Membranes were developed using chemiluminescence (ECL select, Amersham) on an Azure Sapphire imager using the chemiluminescence setting with High Dynamic Range (HDR), auto exposure, and cumulative capture. Membranes were also stained with Coomassie brilliant blue to visualize protein purity.