Mouse anti his antibody

Binding of affinity module alone and of affinity-module-dCK fusion proteins to ectodomain IV of Her2 was detected by performing a protein ELISA as described before [32 (link)]. Briefly, 20 nM mouse anti-His antibody (Invitrogen) was used to capture 20 nM of recombinant human ErbB2/Her2 Fc Chimera (R&D Systems) onto Nunc polystyrene microtiter plate wells (Thermo Fisher Scientific). Fifty nM of the affinity module alone or of the affinity-module-dCK fusion protein were added to the wells, and binding detected with an anti-Myc antibody (9E10) conjugated to biotin (Santa Cruz Biotechnology) and streptavidin-conjugated to horseradish peroxidase (HRP; Abcam). The human Her2 ELISA Kit (Invitrogen) was used to determine the number of Her 2 receptors in cell lines. Briefly, 10⁷ cells were lysed in cell extraction buffer (Invitrogen), diluted to a volume of 100 μL/well, and the copy number of Her2 receptor/ cell calculated according to the manufacturer’s protocol.

Microlon 96-well plates (Corning) were coated overnight with either 2.5 µg ml⁻¹ streptavidin (Jackson ImmunoResearch) or 2.5 μg ml⁻¹ mouse anti-His antibody (Thermo Scientific) in phosphate-buffered saline (PBS) at 50 μl per well. Plates were then washed 4 times with PBS-Tween (0.05%) and blocked with PBS + 3% BSA for 1 h at room temperature. Avi-biotinlylated His-tagged JR-FL E168K SOSIP, Avi-biotinlylated BG505 N332 SOSIP, or Avi-biotinlylated B41 SOSIP protein was added at 1 μg ml⁻¹ in PBS + 1% BSA for 2 h at room temperature. Plates were then washed 4 times with PBS-tween (0.05%) and serially diluted monoclonal antibodies in PBS + 1% BSA were added for 1 h at room temperature (20 µg mL⁻¹ in 4-fold titrations). Plates were then washed 4 times with PBS-tween (0.05%) and alkaline phosphatase-conjugated goat anti-human IgG-Fc (Jackson ImmunoResearch, #109-055-098), was added for 1 h at a 1:1000 dilution (final concentration 0.33 μg ml⁻¹) in PBS + 1% BSA at room temperature. Plates were then washed 4 times with PBS-tween (0.05%) and 1 time with purified H₂O and absorption at 405 nm was measured following addition of phosphatase substrate in alkaline phosphatase buffer. EC₅₀ binding titers were calculated using Graphpad Prism v7.0. All EC₅₀ titers for antigenicity comparisons were measured in two or more independent experiments and were subsequently averaged.

HEK293 cells were seeded in a 6-well plate with approximately 25% density and let grow overnight at 37°C, 5% CO₂. The cells were then transfected using calcium phosphate transfection method. In brief, 0.1 μg Lenti-HTTex1 Q103-EGFP plasmid and differential amounts of Lenti–csgA-6x His plasmid (0, 0.2, or 0.3 μg), were added in each snap-cap tube. Beta-galactosidase cDNA cloned in similar vector was used as control. The total amount of DNA in each transfection mixture was normalized using an empty plasmid backbone. 2 M CaCl₂ was subsequently added (0.12 M final concentration), and the volume of mixture was brought up to 100 μL with ddH₂O. 100 μL 2x HBS was added next dropwise and part of solution was squirted into the rest by pipetting for aeration. Entire volume of each transfection mixture was added to the cells and incubation was done overnight at 37°C, 5% CO₂. The transfected cells were subjected to widefield fluorescence microscopy for quantifying HTTex1 Q103-EGFP aggregates and then harvested for SDS-PAGE and western blotting analyses for HTTex1 Q103-EGFP (probed with PHP2 antibody, 1:1,000 dilution) and CsgA-6x His (probed with mouse anti-His antibody, from Thermo Fisher, 1:5,000 dilution).