2.12. Activity staining of antioxidants (SOD and CAT)
The active levels of all antioxidant enzymes were determined using
non-denaturing PAGE of thyroid extracts using the method described
above.24 In each lane of 10% and 8% non-denaturing
PAGE, a thyroid extract containing 100 µg protein for SOD and catalase
was loaded. Following electrophoresis at 4±2°C, the gels were stained
with substrate specific antioxidant enzymes. The SOD staining solution
contained 2.5 mM NBT, 28 mM riboflavin, and 28 mM TEMED. Gels were
incubated in the dark for 20 minutes before being exposed to fluorescent
light until achromatic SOD bands developed against a dark blue
background. In the case of catalase, gels were first soaked in 0.003%
H2O2 for 10 minutes before being incubated in a staining mixture
containing 2% potassium ferricyanide and 2% ferric chloride. Catalase
bands appeared achromatic against a blue-green background.
Gel densitometry was used to calculate the intensities of all bands
using alpha imager gel documentation software. PAGE was performed 3-4
times in each case, and the mean SD of densitometry values of the bands
as a percentage of the control lane are presented in the result along
with one representative gel photograph.