Dear researchers,

I have some confusion in calculating and expressing the IC50 values for alpha amylase inhibition assay. Please help me clear it.

Here is my protocol

1) 100uL extract and acarbose (concentrations of 100,50, 25, 12.5, 6.25, 3.125, 1.56ug/mL).

2) 100uL amylase enzyme (3U/mL) pH 6.8.

3) Pre-incubate at 37oC for 40min.

4) Add 100uL of 1% starch solution and incubate at 37oC for 10min.

5) Stop the reaction by adding 100uL DNS and boil the tubes at 100oC for 5min

6) Read the absorbance at 540 nm. Calculate the IC50.

My question is for calculating the IC50 values should I consider the concentrations as in the tubes/stocks (100,50, 25, 12.5, 6.25, 3.125, 1.56ug/mL) or should I calculate it to the total reaction mixture (400uL) and then express  per mL (ug/mL). This will decrease my IC50 by four times. My results are matching the literature if I don't consider the dilution factor. Please suggest me the right of doing it.

Thanks

Nimmi