I use the dinitrosalicylic acid (DNS) procedure (Bernfeld, 1955) to assay alpha amylase activity assay in insects and read the absorbance at 540 nm.
I would like to know which amounts of OD are acceptable in this method. I heard that the OD value should be between 0.5 and 1. Is the statement true or not?
Hi, The statement can be fine but you must use both Blank and Negative control. The second one requires pre-boiled sample for at least 30 min.
Best,
Hi Maedeh,
In general, we can adopt this statement as correct, but the answer is not so simple. It is important to understand why it is necessary to take the optical density (absorbance) value in account. Many devices such as spectrophotometers and ELISA readers have a limited capability to give a linear response when the optical density is high. Any device have its own limitation. A good approach is to make a concentration curve (different concentrations of carbohydrate versus obtained OD measured in your device in your condictions). This procedure will give you the concentration from which the absorbance is non-linear. Only values similar to those in the linear part of the curve are acceptable.
A good example of amylase assay can be encountered in the paper: Carbohydrate digestion in Lutzomyia longipalpis’ larvae (Diptera – Psychodidae). Jounal of insect physiology.Volume 58, Issue 10, October 2012, Pages 1314–1324.
Regards,
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