I'm working on potential of plant extract phenolic compound on amylase and glucosidase inhibition.
Currently i am facing a problem on my amylase inhibition assay where i obtain inconsistent inhibition percentage with increasing extract concentration.
I'm using bacillus amylase and phosphate buffer ph6.9 for my assay and my protocol are:
200ul of 2U/ml of enzyme added with 200ul of plant extract which then allowed for incubation in 37degree for 10minutes. Next, further incubation for 3minutes after addition of 200ul of 1%starch. Then, addition of 200ul of DNS reagent and the mixture is boiled for 10minutes. Lastly, 5ml of distilled water is added and the absorbance is read under 540nm absorbance.
I had tried various alternative for example by testing using vast range of concentration, adjusting the temperature of incubation, change solvent for dilution which is from methanol to dmso and extracting new leaf extract. however, the result obtained is still inconsistent.
Hi
DNS method always hse some problems. by the way, i think
* 3 min is a short time for inhibited enzyme to react with starch.
* Measure samples absorbance against distilled water(the blank and original then calculate the difference as the absorbance)
* Do all assays simultaneous, if you want to compare results.
Good Luck.
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