Mouse anti his monoclonal antibody

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done according to the method of Laemmli (Laemmli, 1970 (link)). Proteins were transferred to nitrocellulose membranes, which were then blocked for 2 h with 5% non-fat dry milk dissolved in 1 × TBST buffer (AmericanBio, United States), and then probed with either mouse anti-His monoclonal antibody (1:2,000 dilution; Santa Cruz Biotechnology, United States) or mouse anti-LA3490 polyclonal antibodies (1:2,000 dilution). After washing thrice with TBST, membranes were incubated for 21/2 h with alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) as the secondary antibody (KPL, United States) at a dilution of 1:5,000 in TBST. Blots were developed in 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium solution (BCIP/NBT; KPL, United States).

The cultured mammalian cells were seeded in a 96-well plate at a density of 6.0 × 10⁴ A549 cells per well and 1.25 × 10⁴ J774A.1 cells per well in the cell culture medium for 1 day, respectively. After serum starvation, 1 μg of the purified and quantified MPs was added to the cells with FBS-free RPMI and incubated in an incubator containing 5% CO₂ at 37 °C for 80 min. The cells were washed with PBS-T four times and fixed with PBS-based 4% paraformaldehyde for 30 min. After each well was washed, the cells were blocked with 0.5% casein in PBS-T at 37 °C for 1 h and washed with PBS-T. After being treated with mouse anti-His monoclonal antibody (1:3000 dilution, Santa Cruz Biotechnology, USA) in PBS-T containing 0.5% casein at RT for 1 h, the cells were washed and incubated with HRP-conjugated anti-mouse IgG antibody (1:2000 dilution, Santa Cruz Biotechnology, USA) at RT for 1 h. After each well was washed, 100 μl of TMB-blotting substrate solution (Sigma-Aldrich, USA) was added to the cells, which were then incubated for 15 min to develop a color change. Then, 100 μl of the stop solution (2 M H₂SO₄) was added to stop the reaction. The absorbance of each well was measured using a UVM 340 microplate reader at 450 nm wavelength.