Recently I am working with alpha-amylase inhibition and had some problems with color change.
In my blank, actually I set 2 blanks, the 1st one is water + starch, then heat with DNS for 5 min; the 2nd one is enzyme ( porcine pancreatic alpha amylase) + water, then heat with DNS for 5 min. I thought the color for this 2 blank should be the same, because DNS only works with reducing sugar, but the problem is the color of 1st one was like "pure" yellow, and the 2nd one was like what DNS reagent's color (yellow-orange). I have no idea why it happens and feel confuse what is the right blank for my experiment.
The protocol I used is:
250µL sample + 250µL enzyme / 250µL acarbose + 250µL enzyme (positive control) / 250µL Milli Q water + 250µL enzyme (negative control) / 500µL Milli Q water (1st blank) / 250µL Milli Q water + 250µL enzyme (2nd blank), then mixture incubated for 10 min at 25°C, add 250µL of 1% starch, then mixture incubated for 10 min at 25°C, after that 500µL of DNS reagent was add to terminate the reaction and the mixture was incubated for 5 min at 100 °C, cooled to room temperature and then solution was diluted by 5 ml of Milli Q water. The absorbance was recorded at 540 nm.
Thank you.
Hi Zhujun,
What about the color of starch solution and the enzyme solution. Are they same? We have an experience that sample colors interfere with the test results.
I have same problem here if you got answer please share with us
so, in the first case, you have added starch+dns and heated for 5mins at 100C, starch being a non reducing sugar, it wont get reduced without the amylase being acted upon it. once starch gets reduced it forms maltose units. DNS (yellow colour solour) only reacts with the reduced maltose. so in case 1: you have not added the amylase, hence starch is not reduced to form maltose and dns didnot react with the same to give you final red colour. in case 2: you have added only amylase and DNS. Since there is no starch in this, there is no point of formation of the compound.
hence there is no colour formation at all in the both the cases.
so i suggest you to try amylase+starch+dns+heating.
and by the way why did you cooled down the reaction mixture and diluted and then taken the readings?
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