Data fitting

I have a problem with the data fitting, the calculated fit does not correspond to the measured curve, where you can see a big shift at the dissociation phase. First I thought the problem may arise from the spikes, which are only absent for the two highest concentrations, but present in the rest of the curves, so I tried to fit then only first two curves, hower the problem did not go, here the fitting model is still the same. The amount of immobilized ligand was 129 RU. The initial estimated Kd was around 100 nM, so I went with the concentrations of the analyte from1 uM to 7 nM. For the fitting 1:1 Langmuir model was used.

Hi,

The problem you see is because the interaction is not entirely 1:1 (see www.sprpages.nl/data-fitting/troubleshooting/high-buffer-jump ).
The start of the dissociation is different from the rest since first the weak bound molecules are dissociating and give a fast drop. the the more stronger binding molecules dissociate slower.
Options: check the purity of ligand and analyte. Stay within 0.1 - 10 x KD.

kind regards

Arnoud

Thank you! Do you also have an idea, why do I get spikes for only low concentrations of the analyte, but not for the higher ones? I run this experiment in 2 replicas and got the same picture

Thank you! Do you also have an idea why i get spikes for the low concentration of the analyte but not for the ones with higher concentrations?

Do you man spikes at the start and end of the injection?
Please see www.sprpages.nl/troubleshooting/bulk-and-spikes

Is this helping? Otherwise can you show in a figure what you mean.

Arnoud