I'm not familiar with the T100 Evaluation software but have you tried analyzing the single curves (i.e. not a global fit with all 4 conc) ? I've gotten screwy nonsensical numbers in the past doing a global fit and it has turned out that only one of the concentrations was throwing the whole fit out of line. When I discovered which concentration was askew, I was able to adjust the region selected for fitting and get a more reasonable global fit.
I am measuring the interaction between a protein (coated) and a small inhibitor (200Da) on biacoreT100. To get high responce I immobilized high level of protein 10000RU and got a good resonse of the inhibitor (Rmax 50), which is specific (no unspecific on reference and negative control doesnt bind to the coated flow cell), the analyte concentration are 7-500µM.
the sensorgram looks good, the KD seems to be ok, but the ka is too low (13[1/M*s]!!!).
What am I doing wrong?
I analyzed the curves with 1:1 model with T100 evaluation software,
the ka is 13[1/M*s], the kd is 0,002471[1/s], KD 1,853E-4 and rmax 55, flow 20µl/min, Chi2=2,11. When I simulate the curves I get the similar curves.
The simulation is ok with the exception of the beginning and the end of the injection where the buffer jump is simulated insetad of a fast a- and desociation.
Why do you think the ka is not right (13 [1/M*s] is very slow)? On what basis do you think the KD is OK. Did you other types of experiments to determine this?
You can try to fit without the bufferjump, but it looks like the kinetics are not exactly 1:1.
Also you can try the fitting module of Scrubber when you have that and compare.
Try fitting the kd alone: is it the same?
Yes, the KD ( the IC50 value for this inhibitor ) has been determined in enzyme activity assay to be in this range.
The ka is too low because: the Quality control of biacore T100 says: ka outside the limits that can be measured by the instrument! (I don´t really know what that means, beacause it is actually measured??)
And I think that so slow interaction couldnt cause fast enzyme inactivation?
I tried to fit without buffer jump, but i think now this is not really a problem, but the non-1:1 kinetics, or some unspecific binding on denatured protein part on the chip next to the actual binding.
When I fit kd alone i get values from 7,82e-4 for the highest concentration and 2,92e-3 for the lowest.