Hrp conjugated goat anti mouse igg h l antibody

Animal sera were evaluated for SARS-CoV-2-S-specific IgG antibodies using ELISA. Briefly, ELISA plates (BRANDplates®, immunoGrade, BRAND GMBH + CO KG) were coated with 100 ng of the recombinant ectodomain of SARS-CoV-2 Wu-1 (Sino Biological 40589-V08B1), Delta (Sino Biological 40589-V08B16) or BA.1 Spike protein (Sino Biological 40589-V08H26) per well overnight at 4 °C in 50 μL PBS and then blocked with PBS-T/3% BSA (blocking buffer) for 1 h. Following the procedure as described, bound-specific IgG was detected with an HRP-conjugated goat anti-mouse IgG H&L antibody (ab6789, Abcam) diluted at 1: 10,000 in a blocking buffer. Wu-1-S-specific IgG1e3 mAb (Invivogen, Waltham, MA, USA) and SARS-CoV-2 Spike Antibody, Omicron Reactive, Mouse Mab 40592-MM117 (Sino Biological) were used as positive controls. Color development was performed by the addition of 50 μL of TMB Single Solution (Thermo Fisher Scientific, Carlsbad, CA, USA). After 8 min, the enzyme reaction was stopped with 50 μL of 1 M sulfuric acid per well, and the absorbance was measured in a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The sera were assayed in duplicates, and the antibody titer represents the last reciprocal serum dilution above blank.

After cultivation, normalized number of cells was harvested; cells were disrupted, and protein was extracted using bead beating. After protein extraction, the total extract was boiled with SDS-PAGE sample buffer for 5 min (Green et al. 2012 ). The boiled samples were cooled and subjected to SDS-PAGE. After gel electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF; Bio-Rad) membranes, which were then blocked by 5% (w/v) skim milk solution in Tris-buffered saline containing Tween-20 (TBST) for 1 h. After blocking, the membranes were washed four times with TBST. The membranes were incubated in 1:2000 diluted mouse anti-mCherry monoclonal antibody [1C51] (Abcam, Cambridge, UK) in blocking solution at 25 °C. After 1 h, the primary antibody solution was discarded, and the membranes were incubated in 1:10,000 diluted HRP-conjugated goat anti-mouse IgG H&L antibody (Abcam) for 1 h. The membranes were washed four times with TBST for 10 min, and then subjected to chemiluminescent analysis. The bands were visualized using ECL Western blotting substrate (Promega) and detected using a Chemiluminescence Imaging System (Atto Korea, Daejeon, Korea).