Hrp conjugated goat anti mouse igg h l secondary antibody

Peptide ELISA was performed with synthesized peptides (Genescripts). These peptides were tethered by N-terminal biotinylated linker peptides (biotin-ahx). The RBD9.1 amino acid residues were selected and mutated to alanine and synthesized by Genescripts (Wuhan, China). Here, 50 μl synthesized peptides were added to the streptavidin-coated 384-well plate in duplets to make a final concentration of 5 μg/ml. The plates were incubated for 2 h at room temperature (RT). After washing, the plates were blocked with Protein-Free Blocking Buffer (Pierce) at RT for 1 h and incubated with 50 µl testing serum with 1,000-fold dilution at RT for another 1 h. Reacted human SARS-CoV-2 convalescent serum was detected using ALkaline Phosphatase (ALP)-conjugated Goat F(ab’)2 Anti-Human [IgG (Fab’)2] secondary antibody (Abcam, ab98532, 1:2,000). Reacted mouse serum was detected using horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse IgG H&L secondary antibody (Abcam, ab6789, 1:10,000). Peptide exchange rates (%) = [(A-Blank)]/(P-Blank) × 100, where A is the OD signal of 1,000-fold diluted serum binding to RBD9.1 peptide with single point mutant, and P is the OD signal of 1,000-fold diluted serum binding to RBD9.1 peptide (Positive control).

Protein from the untreated cells (0 h) as well as from those subjected to 100 μM biotin for the indicated times was extracted by the addition of TE buffer (0.1 M TRIS-PO₄, 4% SDS, 4 mM Na-EDTA, 40% glycerol, 0.04% bromophenol blue, and 0.04% β-mercaptoethanol; materials from Sigma-Aldrich), and then the samples were sonicated. In certain experiments, when indicated, the cells were pre-treated with 1 mM 4-PBA, 2 μm MG132, or 10 nM BAF overnight prior to biotin addition. Equal amounts of the protein samples were loaded onto 7.5% polyacrylamide gels. For glycosidase treatments, cell lysates containing 20 μg of protein were subjected to 20 U Endoglycosidase H (Endo H) (Sigma, cat.11088726001) or 20 U PNGase F (Roche, cat.11365185001) according to the manufacturer’s protocols. Blots were probed with anti-ABCG2 (Bxp-21, Abcam, cat. ab3380) or anti-β-actin (Sigma, cat. A1978) primary antibodies, and subsequently developed with HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (Abcam, cat. ab97023). Detection was performed by luminography using Clarity Western ECL Substrate (Bio-Rad, cat. 1705060). For quantification, densitometry was carried out by the ImageJ software.