Competition:Analysis of Results:IC50 Determination

Determination of IC50 Values for the Unlabeled Ligand in Competition Experiments

This page demonstrates how IC50 values are determined from competition experiments using different types of plots.  It is important to go through this page even if you are not doing competition studies.

The IC50 concentration for the unlabeled ligand in a competition experiment is the concentration required to inhibit of the radiolabeled ligand to the specific site by 50%

Although the IC50 value can be determined many times by eye, the most accurate method for determining IC50 values is to use non-linear regression analysis using the equation for a sigmoid plot.

Where Top is the top of the curve
Bottom is the bottom of the curve
Y is the amount bound (either as cpm or as % of control)|
X is the concentration of unlabeled ligand

In the graph shown above the bottom of the curve was zero. 50% of bound was halfway between 1000 cpm and 0 cpm. Estimate 50% bound and the IC50 for the following curves?

1. 

2. 

3. 

Return to # 3 under Overview Analysis of Data

ic50_2.gif (4705 bytes) What point on the Y-axis represents 50% binding of the radioligand to the specific biding site?  What is the IC50?
50% bound: 50 %
Log of IC50 = -8
IC50 = 1 x 10-8 M
IC50= 10 nM
What point on the Y-axis represents 50% binding of the radioligand to the specific biding site?  What is the IC50?
50% bound: 600
      1000 - (1000-200)/2 = 600
Log of IC50 = -9.2
IC50= 6.3 X 10-10 M
IC50 = 0.63 nM

Note on a negative log scale you need to read the axis from right to left rather than from left to right.  That is the Log IC50 point is -9.2 and not -10.8.
What point on the Y-axis represents 50% binding of the radioligand to the specific binding site?   What is the IC50?
 

 50% bound: 575
    &IC50nbsp; 100 - (100-15)/2 = 575
Log of IC50 = -8.7
IC50 = 2 X 10-9 M
IC50 = 2 nM

Note on a negative log scale you need to read the axis from right to left rather than from left to right.  That is the Log IC50 point is -8.7 and not -9.3