Inconsistent Rmax between the fitted curve and fitting report - Forum


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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.
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TOPIC: Inconsistent Rmax between the fitted curve and fitting report

Inconsistent Rmax between the fitted curve and fitting report 28 Oct 2019 18:24 #1

I did a two-state reaction fitting. I saw a good match between the fitted and measured curves. So I suppose the Rmax should be close to the measured values. However, in the fitting report, the Rmax is way beyond the range (3174 vs ~150). Can this be optimized by any experimental settings or this is a problem with the software itself?
Thank you for any suggestions in advance.
Wei
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Inconsistent Rmax between the fitted curve and fitting report 28 Oct 2019 19:46 #2

Judging your sensorgram I would not use a kinetic fitting. The association and dissociation are that fast that they cannot be determined by the algorithm. Basically, what you see is that the curves are scaled by the bulk (RI column). In addition, there is no saturation of your surface. Please take a look at www.sprpages.nl/sensorgram-tutorial/a-curve - equilibrium analysis.

Kind regards
Arnoud

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Inconsistent Rmax between the fitted curve and fitting report 04 Nov 2019 19:00 #3

Arnoud as you said, association and dissociation is too fast to determine the rates. Looking at the sensogram, the dissociation starts even before the analyte injection stops (after 70 seconds the peak declined for all concentrations). i.e., the dissociation starts while passing the analyte itself. Does this plot refers a very week binding? Or it should refer as too fast association/dissociation. What would be the suggested optimizations/precautions for this kind of experiments?

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Inconsistent Rmax between the fitted curve and fitting report 04 Nov 2019 19:39 #4

Hi Charles,
It seems to me that the lowering of the curve during analyte injection is more something of the injection system. It looks very systematic. To check you can inject a buffer sample with some extra salt (e.g. + 25 mM NaCl), The injection curve should rise fast, be flat during the injection and fall fast to baseline indicating that the injection system is performing well.
If not, cleaning of the injection system can do the trick. Always use a high performace injection type (e.g. Kinject).
Kind regards
Arnoud

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