Feb 05, 2025
Amylase detection
- 1Government Institute of Forensic Science Nagpur
Protocol Citation: Hariprasad Paikrao 2025. Amylase detection. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbq27vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 05, 2025
Last Modified: February 05, 2025
Protocol Integer ID: 119586
Abstract
The iodine test is used to test for the presence of starch. Starch turns into an
intense “blue-black” colour upon addition of aqueous solution of iodine, due to
the formation of an intermolecular charge transfer complex.
Starch is a polysaccharide consisting of glucose units joined together by glycosidic
bonds. The amylase in the starch reacts with iodine to form a dark blue complex
while amylopectin develops reddish purple colour. In the presence of amylase
the starch is broken down into saccharides, consequently such colour does not
develop, when iodine is added.
A common configuration of the method in the radial diffusion assay and agar gel
containing starch is prepared. A sample well is created by punching a hole in
the gel and extract the questioned sample is then stained using an iodine
solution. Clear area in the gel indicates amylase activity and the size of the
clear area is proportional to the amount of amylose in the sample.
Quantitative analysis of amylase
Quantitative analysis of amylase
1% agar gel was prepared containing 1% starch
The gel was poured in the petriplates and was allowed to solidify
Gel was punchered by using gel punchers into five different wells
20µl of amylase enzyme was added in the range of 2µl, 4µl, 6µl, 8µl, 10µl from the stock of 1mg/ml of amylase in outer wells and unknown enzyme sample in the centre well
The petriplates were incubated in moist chamber
1% of iodine solution was prepared in ethanol and it was poured on the starch agarose plate on next day
White colour zones (transperent) were observed against dark blue colour background
The diameter of white zone of standard and unknown was measured
A graph was plotted with the concentration versus diameter of zone
The unknown concentration was calculated by using a standard graph.
Amylase detection