Native polyacrylamide gel electrophoresis for isoenzyme profiling

Native–PAGE analysis was carried out on discontinuous polyacrylamide gels (PAGE) with 4.5% polyacrylamide in stacking and with its varying concentrations (10% for SOD and GST, 6% for CAT and 8% for POD). The separation of individual isoenzyme was performed in a vertical gel electrophoretic unit (GeNei, India) by considering the method of Laemmli⁶⁰. A uniform amount (300 mg) of proteins mixed with sample buffer (0.5 M Tris–HCl, pH 6.8) was loaded in each well and proteins were electrophoretically separated at 80 V through stacking gel followed by 120 V in the separating gel at 4 ºC. For visualizing SOD isoenzymes, the gels were immersed in PPB (50 mM; pH 7.8) containing NBT (1.125 mM) in darkness for 20 min and followed by incubation in PPB containing TEMED (28 mM) and riboflavin (28 mM) in dark for 15 min. The gels were then placed in PPB containing mM EDTA (0.1 ml) and exposed to light for 20 min at 25 ºC⁶¹. For POD, gels were immersed in 1 mg ml⁻¹ benzidine and 1 mM H₂O₂ in 0.1 M Tris–acetate buffer (pH 5.0) at 25 ºC till the brown colored bands appeared⁶². For CAT isoenzymes gels were incubated with H₂O₂ (0.01%) for 15 min, rinsed with water, and shaken in freshly prepared solutions each of 0.1% FeCl₃ and K₃Fe(CN)₆ at 25 ºC until the achromatic bands appeared⁶³. For GST isoenzyme staining a reaction mixture containing 0.1 M PPB (pH 6.5), GSH (5.0 mM), CDNB (1.0 mM) and NBT (1 mM) was used and bands were developed by illuminating the gel under 25 µmol photon m⁻² s⁻¹ of photon flux density⁶⁴. Photographs of isoenzymes were captured with digital camera, photo-plate was prepared in CorelDRAW and image quality was enhanced in Photoshop 7.0.