Soluble recombinant HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules display different biochemical properties. Characterization of the native structure and stability of both soluble recombinant HLA-DR*1101 molecules. (a) Immunoprecipitation of HLA-DR*1101-Ig and HLA-DR*1101-Bir molecule with 30 μl of L243-sepharose beads from 1 ml of S2 cells culture supernatant after CuSO4 induction. The lanes contain the following material: 1. sup, 30 μl of culture supernatant before immunoprecipitation; 2. bound, the immunoprecipitated soluble recombinant HLA-DR*1101; and 3. not bound, 30 μl of culture supernatant after immunoprecipitation. Proteins were separated on SDS-page, transferred to filter and revealed with anti his-tag antibody. (b) Densitometric analysis of the protein bands displayed in panel (a), showing the elative efficiency of immunoprecipitation of the two soluble recombinant HLA-DR*1101 proteins with L243-Sepharose beads, as an indirect indicator of the percentage of correctly folded molecule. (c) Size exclusion chromatography of HLA-DR*1101-Ig molecules, after purification of ProtA affinity chromatography. The elution profile of the molecule from a Superdex 200 gel filtration column is shown. Inset shows the dot-blot analysis performed on the protein contained in the indicated elution peaks. Spotted proteins are probed with either anti-His tag antibody, to verify the presence of the HLA-DR*1101 molecule, or L243 mAb (Anti-HLA-DR) to verify the correct conformation of the molecule. (d) Elution profile from Superdex 200 gel filtration column and dot-blot analysis on eluted peaks of HLA-DR11-Bir molecules, performed as described in (c). (e) Calibration profile of the Superdex 200 gel filtration column.