The Use of Reducing Agents

The presence of a reducing agent in experimental solutions is often necessary for some proteins to maintain activity and stability and/or prevent it from forming aggregates. Currently, the most popular reducing agent for proteins is dithiothreitol (DTT). However, DTT is unstable and tends to undergo oxidation in the solution in the presence of air. MicroCal research and customer experiences have showed that even at 0.5 mM concentration of DTT could have an adverse effect on the DSC and ITC baselines even though the solution was degassed under the mild vacuum (27 mm Hg) immediately prior to the experiment. Although we found that purging air from the solution with either nitrogen or argon could minimize the effect of DTT on the calorimetry baseline (see figure 1) This method appears to be impractical for a solution containing detergent or protein, as it will foam upon purging with nitrogen or argon.