What factors besides time affect the rate and efficiency of reduction?
Increasing the temperature will help to accelerate reduction, e.g., 30 minutes at 37°C is roughly equivalent to 1 hour at room temperature. For proteins, the efficiency of reduction may be improved by changing to more acidic or alkaline conditions (pH 3-4 or pH 8-9), which can alter protein conformation sufficiently to render certain disulfides more accessible to the reducing agent. Likewise, the addition of a denaturant (e.g., 3 M guanidine-HCl) or detergent can expose otherwise inaccessible disulfides to the immobilized reducing agent.
