Figure 4
From: Functional recombinant MHC class II molecules and high-throughput peptide-binding assays
Disulphide assisted refolding of MHC class II. Comparison of disulphide assisted refolding with traditional oxidative refolding. (A) Gel shift assay showing that reduction of DRA*0101, DRB5*0101 and DRB1*0401 (by 2-mercaptoethanol (2-ME)) resulted in formation of a slower moving isomer, and suggesting the presence of oxidized isomers in non-reduced samples. (B) Urea denatured β chain (4 μM) was reduced in a titration of dithiotreitol (DTT) and diluted 50 times into refolding buffer pH 8 containing various redox pairs plus denatured non-reduced α chain and 125I labeled (Y)HA306–318. After 24 h incubation, the experiment was analyzed by spun column assay. X-axis: Concentration of DTT used to reduce β chain; Y-axis: percent of offered 125I labelled (Y)Ha306–318 peptide incorporated into peptide-MHC-II complexes. (black square) No redox pair (open circle) Reduced/oxidized glutathione 5/0.5 mM, (black triangle) Reduced/oxidized glutathione 2/2 mM (open square) Reduced/oxidized DTT 5/0.5 mM, (cross) Reduced/oxidized DTT 2/2 mM.