Riboflavin

A native gel-based assay for examining SOD activity was carried out according to a previously described method [48 (link)] with slight modifications. Cells were sonicated in a 50 mM potassium phosphate buffer (pH 7.8). Cell protein (20 µg/lane) was run on a native riboflavin gel comprising a 5% stacking gel (pH 6.8) and a 12% running gel (pH 8.8) at 4 °C. To visualize SOD activity, gels were first soaked in 2.43 mM nitro blue tetrazolium (Wako Pure Chemical Industries, Osaka, Japan) in deionized water for 20 min and then in 56 nM riboflavin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) with 28 mM N,N,N′,N′-tetramethylethylenediamine (Sigma Aldrich, St. Louis, MO, USA) in a 50 mM potassium phosphate buffer (pH 7.8) for 15 min in the dark. Gels were washed with deionized water and illuminated under fluorescent light until clear zones of SOD activity were evident. The images were recorded and MnSOD bands were quantified using the AlphaImager imaging system (Alpha Innotech, San Leandro, CA, USA). MnSOD activity was assessed by examining the band density. The MnSOD activity of vector-only-transfected control cells was normalized to a value of 1.0 and relative MnSOD activities in other cells were calculated. Results were calculated as the mean of the integrated density from five independent runs.