Anti groel

Immunoblotting was performed essentially as described previously (45 (link)). Briefly, proteins were separated by SDS-PAGE on 12% BisTris gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Invitrogen), and incubated with anti-His₆ (Bethyl Laboratories, Inc.), anti-β-lactamase (Abcam), or anti-GroEL (Sigma) primary antibodies followed by an appropriate horseradish peroxidase-conjugated secondary antibody (Sigma). Chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) on an Amersham Biosciences Imager 600 (GE Healthcare).

SepSecS mutant enzymes were resolved either on SDS- or native PA gels and transferred onto Low Fluorescence 0.2 μm PVDF membrane (GE Healthcare). For SDS-PAGE blots either 25 or 10 ng of total protein was loaded onto the gel for probing for either the 6xHis-tag or GroEL, respectively. For native PAGE blots, either 3 μg or 350 ng of total protein was loaded for probing for the 6xHis-tag or GroEL, respectively. The membrane was blocked in blocking buffer (5% nonfat dry milk in PBST, 50 mM phosphate buffer, pH 7.6, 150 mM NaCl and 0.1% Tween-20) for 1 h at room temperature. Subsequently, the membrane was incubated with either an anti-His-tag antibody conjugated with horseradish peroxidase (R&D Systems) or anti-GroEL (Sigma-Aldrich) in blocking buffer overnight at 4 °C. Following primary antibody incubation, the GroEL blot was incubated with an HRP-conjugated goat anti-rabbit antibody (Sigma-Aldrich). Immunoblots were developed by enhanced chemiluminescence using ECL Prime Western Detection Reagent (GE Healthcare) and a Konica Minolta SRX-101A imager with Amersham Hyperfilm ECL (GE Healthcare).

Bacterial cultures were grown to the desired conditions. A volume of cells that represents 1 OD_600 nm were pelleted and resuspended in 100 μl of 1× Laemmli buffer. Samples were stored at –20°C. Protein extracts were subjected to SDS-PAGE separation, transfer to PVDF filter and subsequent immunodetection. As primary antibodies, monoclonal anti-FLAG M2 (Sigma-Aldrich #F1804) 1:2000 for both FinO-3×FLAG and ProQ-3×FLAG detection, as loading control GroEL was detected with anti-GroEL (Sigma-Aldrich, #G6532), anti-mouse (Thermo Fisher, cat# 31430) and anti-rabbit (Thermo Fisher, cat# 31460) conjugated to horseradish peroxidase were used as secondary antibodies for anti-FLAG and anti-GroEL respectively. For detection, ECL™ Prime Western Blotting Detection Reagent (GE Healthcare) served as a substrate.

VirF protein levels were detected by western blot through enhanced chemiluminescence. In brief, equal amount of proteins was extracted from strains grown at OD600 ∼0.6, separated on Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Biorad, #4568126) and transferred onto Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Packs (Biorad, #1704156). The stain-free method was used to obtain the loading control (Colella et al. 2012 (link)). The method is based on the fluorescent detection of tryptophan residues in the protein sequence, as a result of the presence of a trihalo compound in the gel. After protein separation by electrophoresis, each gel was imaged upon exposure to UV-light for 5 min and the same region was selected as loading control for all western blots. Immunodetection was performed as described in Di Martino et al. 2016b using polyclonal halon anti-VirF, anti-FLAG (Sigma, #F1804) and anti-GroEL (Sigma, #A8705) antibodies. Quantification by Western blots were obtained by serial dilution of protein extracts, with the relative amounts calculated from a standard curve. For the protein extracts derived from cultures grown at 30°C, concentrated samples were used to calculate the standard curves.