Peroxidase zymograms obtained by agarose native gel electrophoresis have unmet resolution and completeness.

A recently published method to separate protein isoforms by means of a flat-bed agarose native gel was adapted for identifying simultaneously both acid and basic isoforms of plant peroxidases. These were evidenced by in situ activity staining using alternative substrates for which the isoforms showed specific preference. Such approach allowed the detection of a significantly higher number of horseradish peroxidases than the conventional methods based on sample separation by acrylamide gel and the single bands were clearer to observe. Samples recovered from different plant species and with variable level of purity were successfully analyzed for their peroxidase (POX) isoform pattern. We expect that the innovative electrophoretic methodology illustrated in this work will strongly improve the output of experiments that aim at relating the activity and expression variation of specific enzyme biomarkers with physiological and pathological conditions.