I'm able to detect phenotypic differences among mutant strains lacking one or two catalases, but when I try to detect individual catalase activities by zymography after native PAGE, I'm only detecting one band, corresponding to one of the catalases. The other one has been annotated as a catalase-peroxidase, but I'm not able to detect either the catalase or the peroxidase activity after native PAGE. To develop peroxidase activity I've tried different substrates, including DAB, ABTS, TMB and 3-amino-9-ethylcarbazole. but I only detect the positive control (HRP), regardless the substrate employed. Anyone has a clue what's happening?
I suggest you to incubate the gels in different buffers to get activity.
Use buffers of different pH's. May be the other isozyme have different pH optimum?
Unfortunately, this is the optimal pH for the assay. I've tried the same at other pH's and the result was the detection of the positive control, but with faint bands
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