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

Published online by Cambridge University Press:  03 January 2006

MUKESH K. UPADHYAY
Affiliation:
Mycological Research Laboratory, Department of Biological Science, Rani Durgavati University, Jabalpur, Madhya Pradesh-482001 India. rahulxsharma@yahoo.co.in
RAHUL SHARMA
Affiliation:
Mycological Research Laboratory, Department of Biological Science, Rani Durgavati University, Jabalpur, Madhya Pradesh-482001 India. rahulxsharma@yahoo.co.in
AKHILESH K. PANDEY
Affiliation:
Mycological Research Laboratory, Department of Biological Science, Rani Durgavati University, Jabalpur, Madhya Pradesh-482001 India. rahulxsharma@yahoo.co.in
RAM C. RAJAK
Affiliation:
Mycological Research Laboratory, Department of Biological Science, Rani Durgavati University, Jabalpur, Madhya Pradesh-482001 India. rahulxsharma@yahoo.co.in
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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.

Type
Original Article
Copyright
2005 The British Mycological Society

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