Anti his tag antibody

The promoter regions of the target genes were amplified and inserted upstream of the promoter-less egfp gene in the pQE60 vector. The DH5α strain containing pBAD harbouring tr1 (pBAD-rTr1) or ecxR (pBAD-rEcxR), or the pBAD vector, was transformed with the EGFP fusion constructs. After inducing the recombinant protein expression with 0.2% (w/v) arabinose at 37°C for 8 h, EGFP fluorescence was measured for each sample using a Varioskan Flash (Thermo Fisher Scientific) (excitation 395 nm, emission 509 nm) and normalized against the OD₆₀₀. The recombinant protein expression was confirmed with Western blotting using an anti-His-tag antibody (Sigma, MO, USA).

The purified protein was subjected to SDS-PAGE, followed by protein transfer to a nitrocellulose membrane at 25 V for 2 h. The membrane was washed with TBST buffer (1× Tris-borate-EDTA [TBE], 0.1% Tween 20) and blocked for 1 h in TBST buffer containing 5% (wt/vol) nonfat dried milk. His-tagged proteins were detected using an anti-His tag antibody (Sigma-Aldrich, St. Louis, USA) in combination with a secondary antibody, both diluted in bovine serum albumin (BSA) buffer (1× TBE, 0.1% Tween 20, and 5% BSA). The membrane was washed three times with TBST buffer and twice with TBS buffer (1× TBE). Immunoreactive bands were detected by chemiluminescence using the Pierce ECL Western blotting substrate (Thermo Scientific, Rockford, USA) according to the manufacturer's instructions.

Recombinant CDCs (5 µg/mL) were incubated for 30 min at 37 °C and pH 4.5 or pH 7.4. 400 µl of toxin was mixed with 400 µl of 1% hRBCs on ice for 5 min in Buffer C (35 mM sodium phosphate, 125 mM sodium chloride) at the indicated pH. Cells were recovered by centrifugation (800 g) for 10 min at 4 °C. Cells were washed twice with ice cold PBS, and resuspended in 2X SDS buffer + 1% Triton X-100. Bound CDC was detected using an anti-His-tag antibody (Sigma).

In binding assays using coated mycobacteria, dissociated bacterial preparations (0.5 mg, wet weight) were adsorbed on 96-well microplates (Nunc) in carbonate buffer [15 mM Na₂CO₃, 35 mM NaHCO₃, and 1% (w/v) sodium deoxycholate] overnight at 4°C. Wells were then blocked for 2 hours at 37°C with TBS–2 mM CaCl₂–10% BSA and extensively rinsed with TBS–2 mM CaCl₂–1% BSA–1% Tween 20. sDC-SIGN (2 μg ml⁻¹ in TBS–2 mM CaCl₂–1% BSA) preincubated or not with 100 mM mannose and allowed to react with mycobacteria for 2 hour at RT. Wells were washed, and bound sDC-SIGN was detected using mouse monoclonal anti–His-Tag antibody (Sigma-Aldrich) and HRP-conjugated anti-mouse IgG antibody (Sigma-Aldrich). HRP was detected as above.

Purified protein samples were separated by electrophoresis (10% SDS-PAGE) and electroblotted onto a PVDF membrane using a dry transfer system (2 h, 270 mA). Subsequently, the membrane was blocked in the PBS-T buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.05% [v/v] Tween 20, (pH 7.4)) with 3% skimmed milk for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with a specific primary antibody (Anti-His-tag antibody 1:1000, Sigma-Aldrich, Cat No. SAB1306082) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, (pH 7.4)) with 3% skimmed milk. After three washing cycles with PBS-T with 3% skimmed milk, the membrane was incubated with the secondary antibody (anti-rabbit antibody conjugated with alkaline phosphatase, 1:20,000, Sigma-Aldrich, Cat No. A3687) for 1 h at room temperature. Finally, the membrane was washed three times in PBS-T buffer for 10 min at room temperature for each cycle. X-ray film and Pierce^TM ECL Plus Western Blotting Substrate (Thermo Scientific, Cat No. 32134) have been used to detect a Western blot chemiluminescent signal.

The proteins were separated by SDS-PAGE using 12% resolving gels and transferred to PVDF membrane (0.45 μm pore size; Merck Millipore, Billerica, MA, USA). The membranes were blocked with 1% casein [0.1 M Tris-HCl, pH 8.0, 200 mM NaCl, 1% casein from bovine milk (Sigma-Aldrich)] overnight at 4 °C. Next, the membranes were washed with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) and then incubated with anti-His-tag antibody (Sigma-Aldrich) or negative and positive goose antisera (anti-GPV) for 1 h at room temperature. After probing with an HRP conjugated secondary antibody for 1 h at room temperature, recombinant antigens were detected using SuperSignal West Pico Substrate (Thermo Fisher Scientific).