Mouse anti his antibody

Samples of 8 μM purified wildtype hOGG1, hOGG1_C28A, hOGG1_C241A and hOGG1_C253A were either oxidised or reduced (see above, Oxidising and reducing conditions) and run on a non-reducing 15% SDS-PAGE gel. Gels were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris/HCl pH 8.3 190 mM glycine, 20% ethanol) using a Mini-transblot cell (Biorad) at 300 mA and 4°C for 1 h. Membrane blocking was performed using TBS (50 mM Tris/HCl pH 7.5 150 mM NaCl)-Albumin Fraction V 2.5% (w/v) followed by washing with TBS-T (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.05% (w/v) Tween-20). The membrane was incubated overnight with the primary, mouse anti-His antibody (Sigma-Aldrich) at 4°C (diluted 1000× following manufacturer's instructions), followed by incubation with the secondary goat anti-mouse IgG antibody (diluted 10 000× in TBS). Blots were developed using Pierce™ ECL Western blotting substrate (ThermoFisher Scientific) and protein bands were visualised by chemiluminescence detection in the Vilber imaging system (see above).

For detection of his‐tagged SaxE in bacterial strains, cells were harvested by centrifugation at 10 000 g for 10 min at 4°C. The supernatant was removed and bacterial pellet was resuspended in protein loading buffer (50 mM Tris‐HCl, pH 7.5, 2% SDS, 0.1% bromphenol blue, 10% glycerol). Samples were then boiled for 10 min at 100°C and centrifuged at 10 000 g for 10 min at 4°C. The resulting supernatant was used for SDS‐PAGE. For detection of his‐tagged SyrM from inoculated filter paper discs, 100 μl of sterilized water was used to vortex and wash off bacteria on paper discs in a 1.5‐ml test tube. The resultant bacterial solutions were subjected to centrifugation, and supernatants were used for protein electrophoresis. For protein gel blot assay, proteins were transferred from the gels onto nitrocellulose membranes at 200 mA for 1.5 h. The membrane was blocked by TBST containing 5% milk powder and probed with mouse anti‐His antibody (1:2000; Sigma, Saint Louis, MO, USA). Goat anti‐Mouse‐HRP (1:10 000; Sigma) was used as secondary antibody. Blots were developed with the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Milwaukee, WI, USA).

Overnight culture of wild type PA14, a clpP::Tn or lon::Tn mutant harboring pMMB67EH-higA-His plasmid was sub-cultured in fresh LB broth to an OD₆₀₀ of 0.5, then induced with 1 mM IPTG for 1 h, followed by treatment with 50 μg/ml streptomycin. Bacteria were collected at 0, 0.5, 1, 2, 3, 4, and 5 h, boiled in 1 × SDS loading buffer, then subjected to SDS-PAGE. Proteins was transferred onto a PVDF membrane and incubated with mouse anti-His antibody (1:2000) (Millipore, USA) at room temperature for 1 h. After washing with 1 × phosphate buffered saline (1 × PBS: 274 mM NaCl, 5.4 mM KCl, 20 mM Na₂HPO₄, 4 mM KH₂PO₄, pH 7.4) for four times, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000) (Promega, USA) at room temperature for 1 h. Signals were detected with the ECL-plus kit (Millipore) and visualized with a Bio-Rad molecular imager ChemiDoc™ XRS+.

In order to identify the candidate receptors for visfatin, a binding screen of his-tagged recombinant visfatin against >2500 human membrane proteins, was performed using the Retrogenix Cell Microarray platform (Macclesfield, UK). In brief, binding conditions were optimised for binding of his-tagged recombinant proteins. Expression vectors encoding each of the human membrane proteins were spotted onto glass slides. A HEK293 cell monolayer was cultured over the glass slide, resulting in overexpression of each of the human membrane proteins via reverse transfection. In the primary screen, slides were incubated with 2.5 ug/mL his-tagged visfatin or his-tagged EGF (control), and receptor interactions were detected using a mouse anti-His antibody (Millipore) followed by an AlexaFluor⁶⁴⁷ anti-mouse antibody (Life Technologies). Receptor hits were identified by visual inspection using ImageQuant software (GE Healthcare, Chicago, IL, USA). Following the primary screen, vectors encoding each of the positive hits were sequenced and 2 confirmatory screens performed. The first confirmatory screen used his-tagged ligands followed by AlexaFluor⁶⁴⁷ anti-His detection. The second confirmatory screen was performed using his-tagged ligands attached to AlexaFluor⁶⁴⁷ and anti-His-coated beads (high sensitivity) were used to confirm specificity.

96-well plates were coated overnight with 500 ng of h1x, h2x or TRX in 100 μl TBS per well in a wet chamber at room temperature. To block unspecific binding, wells were blocked with 2% (VHH ELISA) or 3% BSA (TRAIL ELISA) in TBS or PBS for 1 h. The VHHs (E. coli culture supernatants) or Ab-scTRAIL constructs were incubated at different concentration in 200 μl TBS for 2 h with shaking, and washed three times. VHH were detected by mouse anti-His antibody (Millipore) and a secondary anti-mouse, peroxidase conjugate (GE Healthcare). TRAIL signals were detected using anti-TRAIL conjugate and the detection buffer (R&D Systems, Quantikine TRAIL ELISA kit) according to the manufacturer’s protocol.