First of all, i have narrow knowledge about antidiabetic assay due to this is my first enzymatic test and i have no one to refer. I have a method but when i tried to google every each of the method detailed, the result showed thousands of variety. The method i have is very simple but all the journal showed a lot of alpha glucosidase method that i really dont understand and quite complicated (unfamiliar term like: well plates, IC50, using DMSO) .
The method are:
1. Prepare 0.1M of phosphate buffer (pH 6.9)
2. Preprare a 1U of alpha glucosidase in phosphate buffer
3. Prepare 1mM pNPG in phosphate buffer
4. Mix 50uL with 1000uL of alpha glucosidase
5. Incubate at 37oC for 10 minutes
6. Add 500uL of pNPG
7. Add 1000uL of 0.1M Na2CO3 every 5 minutes interval
8. Take reading using UV-Vis at 405nm
The things is, i tried to study about unit of enzyme. But turn out to be quite complicated since it include activity, time, mass and so on. The enzyme i have now is lyophilized powder Bacillus stearothermophilus, >50 units/mg, 250 UN, bought at Sigma-Aldrich (G3651).
Can someone assist me on this? Any suggestion on method modification are welcomed. Also if my method is reliable, i dont get why i need to put Na2CO3 every 5 minutes and for how long. Any explanation on this?
note:
1. phosphate buffer used will are the sodium phosphate not the potassium phosphate buffer.
2. samples are methanol extract of dried herbal leaves
3. final total volume alpha glucosidase needed: 60000uL
4. Sample will be read using UV-Vis by using cuvette
There is a piece of information missing. The alpha-glucosidase enzyme solution should be described by a concentration. A unit (U) is not a concentration. You need to know the enzyme concentration in U/ml of buffer.
For example, suppose the protocol requires you to make an alpha-glucosidase solution at 10 U/ml, and you need 5 ml of it. Then you can calculate how much of the powder to weigh out and dissolve:
5 ml x 10 U/ml = 50 U.
The information on the bottle of enzyme, or in the certificate of analysis for the specific lot number, will tell you the dry enzyme's specific activity in U/mg. The lot number should be on the bottle. The certificate is obtainable on-line from the manufacturer.
Suppose the specific activity is 60 U/mg. Then the amount of powder you need to weigh out for 50 U is:
50 U/(60 U/mg) = 0.83 mg
This is probably too small an amount to weigh, so start by making a more concentrated stock solution and diluting it to the desired concentration, or make a larger volume of the solution.
I think the purpose of the Na2CO3 is to quench the reaction by raising the pH to one at which the enzyme is inactive. The idea is to run several identical reactions simultaneously, and quench one of them every 5 minutes. At the end, you can measure the absorbance of all the reactions, which differ in reaction time by 5 minutes. Then you can plot a graph of absorbance versus reaction time. This is called a time course or progress curve. From it, you can then measure the initial rate of the reaction, which is the slope of the linear part of the progress curve at the beginning.
Since you are just starting out, see if you can get the book "Biochemical Calculations" by I. Segel.
Adam B Shapiro I am so sorry for my bad. The concentration needed is 1 U/ml.
So let say i needed 60 ml of enzyme at concentration 1 U/ml, the enzyme activity is 50 U/mg.
the amount of powder needed to weight is:
60 U/(50 U/mg) = 1.2 mg
Please confirm if my understanding is correct, to prepare 1U/ml of enzyme, i have to weigh 1.2 mg and add 60 ml of buffer?
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