Forum :: Determination of Ka and Kd


Welcome, Guest
Username: Password: Remember me
This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.

TOPIC: Determination of Ka and Kd

Determination of Ka and Kd 25 Feb 2016 11:02 #1

Hi Arnoud,

I am new to SPR, My question is that how to calculate kinetic parameters Ka and Kd (from sensorgram) and affinity parameters KA and KD for Straptavidin-Biotin interaction? What are the steps I have to go through?

Determination of Ka and Kd 25 Feb 2016 19:36 #2

Hi John,

First of all some definitions about how to call and write the kinetic constants.

Constants.png


Note that the k is in italic and the subscripts with the rate constants are small letters (not italic) and with the equilibrium dissociation constant it is a capital D. The relation between these three is:

KDcalculation.png


We are all aware that there are many different ways to describe these constants but this is the only correct notation. In addition, do not use the KA since it wil confuse your results.

When you have a curve and you fit this curve with a 1:1 langmuir model, you will get the ka and kd as fitted parameters and you can calculate the KD as described above.

I am not sure what you mean with a streptavidin-biotin interaction, but since this interaction is highly stable I would not try to measure it because the dissociation rate is outside the instrument specification.

If you want more information, please start with reading the kinetics section on the SPR-pages (or buy the book of which a new version is available at www.sprpages.nl/contact-spr-pages/sprpages-book).

Kind regards

Arnoud
Last Edit: 25 Feb 2016 19:40 by Arnoud.

Determination of Ka and Kd 27 Feb 2016 05:03 #3

Thank you, this book has all the information about curve fitting ?? for determining Kd and Bmax, should i consider whole sensorgram or just baseline to association phase, thats what i am confused about.

Determination of Ka and Kd 28 Feb 2016 09:52 #4

Hi John,

The book contains all the information to fit sensorgrams. But if you want to learn some curve fitting concepts you can also read the curve fitting guide at (www.graphpad.com/data-analysis-resource-center/). For a better understanding of the sensorgrams, follow the Sensorgrams tutorial at the SPRpages.

To answer your question. To calculate the KD or kd (you confused the symbols) and the Rmax (the maximal response) the complete sensorgram should be used, preferable a set of curves with different analyte concentration in order to get a robust result.

Kind regards

Arnoud

Determination of Ka and Kd 02 Mar 2016 06:43 #5

Is the curve fitting possible in origin pro8 software for determining these association and dissociation constant??

Thank you

Determination of Ka and Kd 02 Mar 2016 08:26 #6

I am not familiar with this software. Probably you have to define the fitting model yourself.

Arnoud
Moderators: Arnoud