Optimization of the gp96/MART-1 complexing reaction. (A) For the initial complexing reaction, previously published complexing conditions were employed (14). The gp96 concentration was 100 µg/ml. Gp96 and MART-1 peptides were coincubated in a 0.7 M sodium phosphate buffer for 10 min at 50°C with a molar ratio of gp96 to peptide of 1:50. To optimize this complexing reaction, molar ratios of gp96 to MART-1 from 1:10 to 1:400 were tested. The optimal molar ratio was determined to be 1:200. Subsequent experiments used the optimal condition from the previous experiments. (B) A gp96 concentration of 300 µg/ml (tested range: 100 to 400 µg/ml) increased the complexing effectiveness compared with 100 µg/ml. (C) When the reaction time was increased (tested range: 5 to 60 min), the complexing efficiency also gradually increased without reaching an equilibrium. However, to avoid protein degradation and/or peptide aggregation, the complexing time in further experiments was restricted to 30 min. (D) Influence of the complexing temperature on the complexing efficiency: At 50°C, 1.7 ng of the MART-1 peptide was complexed to 1 µg of gp96, which is a 10-fold increase compared with the complexing efficiency published previously (14). (E) Reduced complexing efficiency with increasing DMSO percentage during the complexing reaction: Because of the hydrophobicity of the MART-1 peptide, the lowest possible value of DMSO was about 20%. (F) Testing the sodium concentration of the reaction buffer in a range from 0.35 M to 2.8 M did not reveal any improvement, thus the 0.7 M sodium concentration was used in subsequent experiments.