I use Langmuir 1:1 binding model to evaluate kinetics and affinity parameters for a very strong protein-protein interaction. My problem is that the regeneration is never complete so every time I inject new analyte concentration I´m offering a slightly different ligand surface. Is in such case "allowed" to fit Rmax local in the Langmuir 1:1 binding model?
My curves are better fitted and I believe values obtained are more reliable.
Maybe you want to look at the following publications.
If you fit Rmax locally you should be critical at the results: The Rmax between the curves should not differ to much.
Do you run duplicates or even better try to inject a triplicate to investigate the changes in baseline? This will give you an idea of how 'bad' your regeneration is.
Anal Biochem. 2002 Jul 15;306(2):228-36.
Compensation for loss of ligand activity in surface plasmon resonance experiments.
Ober RJ, Ward ES.
Anal Biochem. 1998 Aug 1;261(2):203-10.
Interpreting kinetic rate constants from optical biosensor data recorded on a decaying surface.
Joss L, Morton TA, Doyle ML, Myszka DG.
I´m injecting in each measurement every third or fourth cycle the same positive control sample, I think thats quite good control, and I can see from cycle to cycle almost identical decrease in binding. I´m also checking the baseline and every next cycle is my baseline higher, whereas the RUs which are "unregeneratable" are each cycle similar high, whereas they are only slight influenced by my analyte concentration.
I will go through the publications you are kindly proposing!