Thermal shift assay

LZ-ELBM HLA-B molecules were provided by the NIH tetramer core facility. Peptide-deficient conformers of molecules were prepared by incubation with PreScission protease or thrombin overnight at 25°C. Cleaved fragments were removed by centrifuging the sample in a 0.5 ml Amicon Ultra filter device for 30 min at 13,000 rpm, 4°C. Native-PAGE and SDS-PAGE gels were both run to verify that the cleavage was efficient and HLA-B molecules became peptide-deficient. Peptide exchanges were performed by incubating HLA-B molecules with high affinity peptides together with PreScission protease or thrombin overnight at 25°C. Thermal shift assays were undertaken as previously described (Del Cid et al., 2010; Huynh and Partch, 2015). HLA-B molecules (8 µM) were incubated in buffer (PBS, pH7.4) and 1 × Sypro Orange Stain (Invitrogen) in a total reaction volume of 20 µl. Thermal scans were performed using an ABI PRISM 7900HT Sequence Detection System with temperature increments of 1°C. Fluorescence emission was measured at ROX channel. Fluorescence was normalized within wells as percent maximum fluorescence and plotted against the sample temperature.