Hi, i am doing some study on Inhibition assay for α-glucosidase activity of a plant extract ,
here the reagents i am using
α-glucosidase( 0.02 unit, i.e 5 micro liter)
PNPG 5mm(50 micro liter)
Na2Co3 0.2 m (160 micro liter)
phosphate Buffer pH 6.8
and extract it 10-200 micro liter from (1 mg/ml stock)
but i am not getting any color change between blank and extract.
all are coming yellow and reading almost same with blank.
i have changed the incubation time and morality of Na2Co3 and PNPG but no result.
If some one has done some other method please guide me.
Your reagents and values seem fine. But you could try 10 micro litres α-glucosidase, 20 micro-litres of 1mM PNPG, 50 micro-litres Na2CO3 (0.1 M), 50 micro-litres phosphate buffer (50mM, pH 6.8), and 20 micro-litres plant extract. Use pre-incubation time 5 minutes at 37C then add PNPG and incubate for 30 more minutes before stopping the reaction. You can place in boiling water then cool. I recommend obtaining brand new reagents in case there is a problem there :-)
Hi Andrew,
What is your recommendation unit concentration for the enzyme?
Below was the method I used but I could'nt get the IC50 of acarbose....
50 micro-litres phosphate buffer (30mM, pH 6.8)
10 micro litres α-glucosidase (0.02 U/well),
10 micro-litres acarbose (1.55 mM),
50 micro-litres of PNPG (1mM)
Pre-incubation time 10 minutes at 37C then add PNPG and incubate for 30 more minutes before reading.
Please advise me regarding this matter.
Thank you...
Hi Abdullah,
you can take 1unit/ml concentration and from there you can fix your required volume for the reaction. and the rest of your reagent looks okay but if you make PNPG 3mM that will be better. hope this will solve your problem.
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