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.