Fluorophore conjugated sav

Here, we generated different multivalent formulations of a given pMHC specificity to enable comparative analysis. To ascribe the observed differences to the multimerization scaffold, all the multivalent pMHC formulations (tetramer, dextramer, and spheromer) were made using the same stock of purified MHC molecules. The pMHC tetramers were generated as described previously (63 (link)). Briefly, fluorophore-conjugated SAv (Invitrogen) was added to each pMHC monomer incrementally to achieve a 4:1 (pMHC:SAv) molar ratio. Next, SAv agarose was added to each tetramer for quenching any unbound, biotinylated pMHC. After filtration, biotinylated agarose beads were added to remove any unsaturated SAv molecules. The protein was filtered and stored at 4°C until further use. We also used a previously described protocol for generating the pMHC dextramers (19 (link)). The biotinylated pMHC molecules were incubated with fluorophore-conjugated SAv (Invitrogen) at a molar ratio of ~3.5:1 (pMHC:SAv) for 30 min at room temperature. To this mixture, biotin-dextran (M_W = 70 kDa, Thermo Fisher Scientific) was added at a molar ratio of ~30:1 (pMHC:dextran) and incubated further for another 30 min at room temperature. The spheromer assembly has already been described above.