I am doing amylase activity assay using DNSA method I add 0.5 ml substrate + 0.5 ml enzyme then I leave them in water bath at 50oC for 30min after that i stopped the reacation with 2 ml DNSA then go to boiling for 5 min but I didn’t get any changes in color although the starch was degraded in liquid medium !
7/13/23
Dear Nouran,
How did you determine that the starch was degraded by the amylase? Also, what was the potency of the a-amylase and the amount added to the activity assay? If the potency and amount added were too low, it's possible that the amount of reducing sugar released from the starch in 30 min was below the DNSA limit of detection.
You didn't mention this in your question, but did you include a reducing sugar control/standard (e.g., glucose, maltose, etc.) in the DNSA reaction? If you did, but saw no color change w/ the DNSA, then there is something wrong w/ the DNSA. If you did see a color change, then you may be using insufficient amylase or insufficient time for starch hydrolysis. Also, what did you use as a buffer for the assay, e.g, pH?
I hope this information helps you.
Bill Colonna Iowa State University, Ames, Iowa, USA [email protected]
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