An improved zymographic method for detection of amylolytic enzymes of fungi on polyacrylamide gels

Abstract

Zymography is an electrophoretic technique by which enzyme activity can be visualized directly on a polyacrylamide gel as discrete bands. A modified, more rapid technique for amylase zymography is described and compared with previously published methods. Whereas previous methods are based on 0.1 M acetate buffer as substrate buffer, our method utilizes 50mM Tris buffer containing Ca²⁺, Na⁺, NaN³ and Triton X-100 which helps rapid hydrolysis of the starch and stabilization of the enzyme. The staining procedure, previously requiring overnight incubation of the gel in iodine solution at 4°C, has been reduced to 5 min at room temperature. Both methods gave rise to comparable levels of enzyme activity on polyacrylamide gels. Our modified method requires 8 h to complete the whole zymographical procedure instead of 18-20 h as in previous methods.