Purification of the gp96/MART-1 complex. (A) A Coomassie-stained SDS gel on the bottom and its gel autoradiography on the top show the gp96/MART-1 complex reconstituted with iodine-125-labeled MART-1 peptides before purification, after repeated gel filtration, and after additional ultrafiltration. Lane 1: 1 µg unpurified gp96/MART-1 complex with approx. 300 ng free peptides on the dye front. Lane 2: protein standard. Lane 3: 0.5 µg of the complex (0.5 µg for technical reasons) after repeated gel filtration (Sephadex G-25) with a 96% reduction in the free peptides, down to 12 ng. Lane 4: 1 µg of the complex after repeated ultrafiltration (membrane with 30-kDa cut-off) at a 1:10⁸ dilution. The amount of uncomplexed peptides was reduced by more than 99.9% to 0.25 ng. (B) The absolute amounts of free and complexed MART-1 peptides during the purification process are shown.