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Hill model and Kd determination
- Leo1212
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3 years 5 months ago #1
by Leo1212
Hill model and Kd determination was created by Leo1212
Dear all,
I am a total beginner in the SPR so sorry if my questions are basic.
We are studying complexation of small cationic molecules by immobilized DNA molecules of different size (20 molecules for DNA 10bp). Since it's not 1:1 interaction we have used a Hill model. We didn't managed to mesure kinetics because kon and koff rates were extremely rapid. I would like to know if concentration at 50% of Bmax is the Kd or it's mostly EC50 since we cannot measure kon/koff?
Thank you in advance
I am a total beginner in the SPR so sorry if my questions are basic.
We are studying complexation of small cationic molecules by immobilized DNA molecules of different size (20 molecules for DNA 10bp). Since it's not 1:1 interaction we have used a Hill model. We didn't managed to mesure kinetics because kon and koff rates were extremely rapid. I would like to know if concentration at 50% of Bmax is the Kd or it's mostly EC50 since we cannot measure kon/koff?
Thank you in advance
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- Arnoud
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3 years 5 months ago #2
by Arnoud
Replied by Arnoud on topic Hill model and Kd determination
Hi,
I am not sure that the hill model is appropriate for what you want to achieve: en.wikipedia.org/wiki/Hill_equation_(biochemistry) .
Since you have a fast on and off rate, equilibrium will be reached very fast. The response at equilibrium is a measure for binding as long as you can saturate the ligand ( www.sprpages.nl/sensorgram-tutorial/a-curve ). Even when the interaction is not 1:1 this will work. Using the equilibrium analysis ( www.sprpages.nl/data-fitting/models/equilibrium-analysis ) you can determine the overall KD of the system.
Keep in mind that the IC50/EC50 is not the same as KD. When KD is << Atot the EC50 provides no information about the KD. When the KD >> Atot the EC50 and the KD will be close. In general, the EC50 overestimates the KD.
kind regards
Arnoud
I am not sure that the hill model is appropriate for what you want to achieve: en.wikipedia.org/wiki/Hill_equation_(biochemistry) .
Since you have a fast on and off rate, equilibrium will be reached very fast. The response at equilibrium is a measure for binding as long as you can saturate the ligand ( www.sprpages.nl/sensorgram-tutorial/a-curve ). Even when the interaction is not 1:1 this will work. Using the equilibrium analysis ( www.sprpages.nl/data-fitting/models/equilibrium-analysis ) you can determine the overall KD of the system.
Keep in mind that the IC50/EC50 is not the same as KD. When KD is << Atot the EC50 provides no information about the KD. When the KD >> Atot the EC50 and the KD will be close. In general, the EC50 overestimates the KD.
kind regards
Arnoud
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