| Question |
Comment |
| Did you go out to a long enough time point? |
Dissociation and association data should plateau, so the data obtained at the last few time points should be indistinguishable. |
| Is the value of kon reasonable? |
The association rate constant, kon, depends largely on diffusion so is similar for many ligands. Expect a result of about 108 M-1 min-1 |
| Is the value of koff reasonable? |
If the koff is greater than 1 min-1, the ligand has a low affinity for the receptor, dissociation will occur while you are separating bound and free ligands, and you'll have a hard time obtaining quality data. If koff is less than 0.001 min-1 , you'll have a difficult time obtaining equilibrium as the half-time of dissociation will be greater than 10 hours! Even if you wait that long, other reactions may occur that ruin the experiment. |
| Are the standard errors too large? Are the confidence intervals too wide. |
Examine the SE and the confidence intervals to see how much confidence you have in the rate constants. |
| Does only a tiny fraction of radioligand bind to the receptors. |
The standard analyses of association experiments assume that the concentration of free radioligand is constant during the experiment. This will be approximately true only if a tiny fraction of the added radioligand binds to the receptors. Compare the maximum total binding in cpm to the amount of added radioligand in cpm. If that ratio exceeds 10% or so, you should revise your experimental protocol. |
