First of all I would like to thank you for all your suggestions which have helped me in solving the issues. Now am facing a new problem in single cycle kinetics. I am using CM5 Chip and doing a capture method. Instrument used is BIACORE T200.
I am using a high affinity molecule and hence the dissociation is too slow. When i put a set of 3 runs of the same molecule my ka is constantly increasing due to which my KD is not consistent.
You suggestions will be highly appreciated.
What do you mean with three runs?
- three analyte injections after each other (single cycle kinetics)
- three different single cycle kinetic runs
I agree that if you do three separate single cycle kinetic runs, the fitted values between the runs should be comparable. You mention that the dissociation is very slow. To have any reliable kd results, the dissociation time should long enough to have at least 5% dissociation in the response level (www.sprpages.nl/kinetics/dissociation). It is beneficial to invest some time in the dissociation; you only need to wait a little longer.
The other thing is the capture method. Although the kinetics should not change when there is more or less ligand on the sensor chip, sometimes this is not the case (e.g. with mass transport). Make sure you capture the same amount of ligand and that the capture molecule is fully regenerated (empty) after a run.
One suggestion when analysing the curves. If you have three identical runs, you can analyse them globally so that you get one value for each of the kinetic parameters (ka, kd, KD). It depends on the reproducibility of the capture method if you should have the Rmax globally or locally. In general the global parameters are more robust.
How much is the difference in ka between the three runs?