There is no absolute rule that say which method of curve fitting is theoretical best. It should just fit the data well.

I think you have used the inhibition with fixed slope function in Graphpad and the AAT Bioquest appears to use the same function but with variable slope (also available in Graphpad, I get the same number with that function).

Your dataset lacks a few measurement at higher than 50% inhibition. A datapoint with 70-90% inhibition would define the curve better. You can help this be defining the min and max of the function. In this case we know theoretically that the inhibition is 100 % at very high concentration and 0% in absence of the substance. If I set the min and max to 0 and 100 you get a better fit and estimate. I got 209 and 257 µg/ml, with and without the fixed slope. I think the variable slope result of 257 µg/ml is a good result as it fits the measured data very well.

It's better to use GraphPad Prism for calculating the IC50 values. You can calculate both absolute and relative IC50 values with your results.

Thank you sir for your answer. I'll try to follow it.

Dear Ms. Pushpa Reddy,

Ideally, if you have linear relation between the two factors, then there should be much variation in the IC50 values, Irrespective of which software you use the equation used for linear regression is same (y=mx+c), and hence you should get almost same values.

I have analyzed the data you have provided, please find the attachment, you get IC50 value of 178.6 for your data, in both MS-Excel and Graph Pad Prism.

Hope it is useful

Regards

Vishwanatha