Figure 1.
Interaction between recombinant, soluble TPN and HLA-B*0801. (A) TPN (10 μg) and HLA-B*0801 (10 μg) (1:1 molar ratio) were incubated on ice in 20 mM Tris, 150 mM NaCl, and 10% glycerol (pH 7.5) for 30 min. Samples were analyzed on a native gel (8%): lane 1, TPN; lane 2, peptide-filled HLA-B*0801; lane 3, peptide-deficient HLA-B*0801; lane 4, mixture of TPN and peptide-filled HLA-B*0801; and lane 5, mixture of TPN and peptide-deficient HLA-B*0801. The gel was run at 4°C in 25 mM Tris and 200 mM glycine (pH 8.3). (B) At time=0, HLA-B*0801 (40 nM) loaded with EIYK*RWIIL (○) was added under stirring to a 1-cm cuvette containing 1000-fold molar excess of the nontagged peptide and 100-fold molar excess of β2m in 20 mM Hepes and 150 mM NaCl (pH 7.5). Peptide dissociation was monitored at 20°C (excitation and emission wavelengths were 495 and 524 nm, respectively). The experiment was repeated in the presence of TPN (400 nM) (•), which was pre-mixed in the buffer before adding HLA-B*0801. Data points were collected for 60 h.