The dissolved MHC-I proteins were purified by Ni 2 +/IDA metal chelating affinity column chromatography followed by Q-Sepharose ion exchange column chromatography, hydrophobic interaction chromatography, and eventually by Superdex-200 size exclusion chromatography. Fractions containing MHC-I heavy chain molecules were identified by A280 absorbance and SDS-PAGE and pooled. Throughout purification and storage, the MHC-I heavy chain proteins were dissolved in 8 M urea to keep them denatured. Note that the MHC-I heavy chain proteins at no time were exposed to reducing conditions. This allowed purification of highly active pre-oxidized moieties as previously described (Ostergaard et al. 2001) . Protein concentrations were determined by bicinchoninic acid assay. The degree of biotinylation (usually >95%) was determined by a gel-shift assay (Leisner et al. 2008 ). The pre-oxidized, denatured proteins were stored at −20°C in Tris-buffered 8 M urea.