Figure 4.

Determination of the in vitro redox status of Cys groups in AnnAt1-His(6) protein. A schematic diagram showing the step-by-step procedure for this experiment is shown. Ten micrograms of AnnAt1-His(6) protein was analyzed. N-ethylmaleimide (NEM) was used to block free SH groups. Dithiothreitol (DTT) was the chemical reducing agent used. Protein SHs were labeled with MPB. After electrophoresis and blotting, one of the replicate filters was probed with avidin conjugated with horseradish peroxidase. To confirm equal loading, the second filter was immunoprobed with anti-annexin antibody (data not shown). This experiment was performed three times and gave comparable results.