Anti human igg fc antibody

Epidermal growth factor receptor (EGFR) was stained as described [32 (link)], 2.5 × 10⁴ cells were seeded in 12-well plate in DMEM 10%. The next day, cells were washed in PBS and permeabilized with 0.3% Triton X-100 for 10 min. Cells were incubated with primary antibodies, Goat anti-EGFR (1:1000, Dako, Santa Clara, CA, USA), and Rabbit anti-phospho-mTOR (1:1000, cell signaling) for 24 h at 4 °C. Cetuximab (CTX) was detected using anti-human IgG Fc antibody (1:1000; R&D systems) for 24 h at 4 °C. After the wash step with PBS1X, cells were incubated for 1 h at room temperature with Alexa 560 labeled anti-goat secondary antibodies (1:400, Invitrogen). Sections were then stained with Hoechst (Thermo Scientific, Waltham, MA, USA) and washed with PBS three times. The cells were analyzed using fluorescence microscopy (Olympus, Fluoview FV10, Shinjuku, Tokyo, Japan) at 600× magnification.

The SPR assay was performed in a BIAcore T200 instrument (GE Healthcare, Piscataway, NJ, USA). An anti-human IgG Fc antibody (R&D Systems, Minneapolis, USA, Cat. No.: MAB110) was immobilized on the surface of a CM5 sensor chip (GE Healthcare Piscataway, NJ, USA, Cat No.: BR100530) at 125 RU. Anti-SARS-CoV-2 antibodies were flown for 60 s at a rate of 10 µL/min. After capturing the antibodies, RBD-WT, RBD-DT or RBD-O, in concentrations ranging from 7.8 to 125 nM, were flown over the chip for 120 s at a rate of 10 μL/min followed by 5 min of dissociation time. The running buffer was HBS-Ep+ (Cytiva, Piscataway, NJ, USA, Cat. No.: BR100669), which contains 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20 at pH 7.4. Regeneration to the baseline was achieved by injections of Glycine-HCl 10 mM (pH 1.5). Association (kon) and dissociation (koff) rate constants were calculated by fitting the raw data to a 1:1 model (Langmuir) using BIAevaluation 3.0 software.