Forum :: Kinetic model


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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.

TOPIC: Kinetic model

Kinetic model 14 Oct 2015 05:37 #1

Hi All,

I have a protein which binds to the DNA either as a homodimer or monomer. There are two protein binding sites on the DNA spaced 16 basepairs apart. Both protein binding sites on the DNA differ by only 5% in sequence. In my experiment DNA was immobilized on an SA chip and varying concentrations of protein were passed onto the chip.
My question
1) Should I fit the curves to 1:1 Langmuir binding because the two binding sites on DNA are similar and the two binding sites are present on the same DNA molecule which was immobilized?
2) Should I go with fitting to Hetergeneous lignad-Parallel reactions because it has two binding sites?

Any other suggestions on curve fitting based on the given condition..

Thank You
Last Edit: 14 Oct 2015 05:49 by Rajasree.

Kinetic model 15 Oct 2015 08:58 #2

Hi,

What will happen upon analyte injection will depend on the relative kinetics between the two binding sites and if the binding to one of the site will sterically hinder the second site.

I would start with one-to-one assuming that the binding differences are not that great and ingnoring the steric hindrance. That said, the first fitting will reveal if the kinetics is 1:1 or not.

But if you use the heterogenoues ligand/parallel it will fit better since you have two sets of parameters.

If you want to separate the two binding sites I would suggest to make DNA with either of the binding sites. If you just want to have a dissociation constant for the interaction you can try to do some equilibrium experiments because that will avarage all the interaction kinetics etc.

Can you show a fitting of both models?

Regards

Arnoud
Last Edit: 15 Oct 2015 08:59 by Arnoud. Reason: typo's
Moderators: Arnoud