Rat isotype control igg

To investigate the success of peptide/MHC biotinylation, a 96-well high protein binding plate (Corning) was first coated overnight at 4 °C with 4 μg/ml streptavidin (Promega) in 0.1 M sodium carbonate and then blocked (3% BSA/PBS) for 1 hr at RT. Biotinylated protein samples were diluted in block and added to wells at various dilutions and incubated at RT for 1 hr. Wells were washed extensively with PBST and then exposed to 4 μg/ml of either a MHC class I-reactive (clone M1/42; Biolegend) or rat isotype control IgG (Biolegend) antibody in block. In separate wells, a positive control biotinylated irrelevant rat antibody (Biolegend) was incorporated to validate the extent of streptavidin binding. The plate was again washed with PBST and relevant wells provided a goat anti-rat HRP (Thermo Scientific Fisher) or goat anti-mouse FcγR-specific HRP (Jackson ImmunoResearch, West Grove, PA) antibody in block for 1 hr at RT. Wells were washed with PBST and developed for 5 min after the addition of 200 μl 1-Step Ultra TMB (Thermo Scientific Fisher). Reactions were terminated with 100 μl TMB stop solution (KPL), and the absorbance immediately read at 450 nm using a Cytation 5 Imaging Reader (Biotek, Winooski, VT).