When checked in enzymatic assay, a compound with IC50 37 nM showed a KD value of 20 µM against ITC. I would like to know how can this discrepancy be explained?
Thank you in advance.
3 magnitudes of difference is uncommon, however, IC50 and Kd are completely different parameters. IC50 describes at what concentration the response you are observing, in this case your enzymatic reaction rate, is halved. It may be susceptible to substrate, enzyme, coenzyme concentrations, buffer (e.g pH, ionic strength, presence of additives), or maybe your compound is interacting with your substrate instead of actually binding the active site of your target protein. On the other hand, Kd is calculated based on the equilibrated system and should be in principle transferable between various techniques (although it is tricky in practice). I'd suggest going for calculating Michaelis–Menten kinetics and Ki investigation that would rule out some of the aforementioned parameters.
Is it possible that the ligand is binding slowly to the protein and takes time to reach the maximum inhibitory concentration? John Carter Don Kaiser Jacek Plewka
KD = ([A]*[B])/[AB] for a 1:1 complex. For an ITC experiment, you can know the initial concentrations of A & B and determine the concentration of AB experimentally.
For an ELISA, you don't know the concentration of whichever reagent is plated, call that one A. The [A] is not in general the concentration added to the wells during the "plating" stage, since there will be some equilibrium between the solution phase and plate well-bound phase.
Surface plasmon resonance (SPR) is a similar surface-binding experiment to ELISA, and it can estimate the mass of A bound to the chip. However, this estimate would not include the proportion of A orientated in the direction conducive to binding B, and I would still accept the solution-phase binding of ITC over it. If molecule B is bulky, you can expect steric issues as well with any surface binding which will create a distribution of KD's.
I found but did not read this paper. Maybe it will have something useful for you. If it does, please update this thread with what you've learned for the benefit of future generations. Article Titration ELISA as a Method to Determine the Dissociation Co...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line