Single Cell Kinetics for Binding Analysis of Small molecules

Dear Aroud
I am trying to use Series S Protein A chip for capturing Fc-tagged Ligand and then examine the binding of small molecules at 5 different concentrations. How can I set the working protocol. Do I need to regenerate the surface by removing ligand-analyte complex after each concentration of analyte binding and then fresh recapturing Fc-Tagged ligand and testing for other concentrations one by one? How can I set the working protocol? Is there any way that I do ligand capture for once and then run 5 concentrations of analyte at a time on that?
Thanks
Joydeb

Dear Joydeb,

You can capture the Fc-tagged ligand once with the Protein A chip and inject five subsequent higher analyte concentrations in one go. This is called the kinetic titration ( www.sprpages.nl/experiments/strategy ). When the interaction is not strong you can wait for a full dissociation en run the next cycle. or try a mild regeneration.

When the ligand-analyte interaction is strong a full regeneration of analyte and Fc-ligand is needed and for the next cycle you have to capture fresh Fc-ligand.

The Biacore T200 has a wizard for this type of experiments.

Good luck!
Arnoud

Thank you Arnoud.
I found the Biacore Protocol for Single Cycle Kinetics but could not find the way how to define the Cycles, specially for capturing the Fc-Tagged ligand. In LKW single cycle kinetics method wizard, there is defined steps of Start up, sample and solvent correction.
My small molecules are soluble in 2% DMSO. So I am using HBS-EP+ 1X with 2% DMSO as running buffer all through the experiment. Is that OK?
Can I perform the whole experiment by using Manual Run option or I must follow a written protocol?
Thank you once again.
Sincerely
Joydeb

Hi Joydeb,
I looked quick for a protocol. It is probably easier to use the kinetics wizard do define a multicycle method with the capture and save that. Then open it in the method editor and change the sample injection to 'single cycle'. Then you can fill in the five analyte concentrations. In addition, include some cycles with five buffer injections to compensate for ligand dissociation.

You cannot do the single cycle kinetics by hand and then analyze it because the analysis software expects certain key-points/key-words.

Arnoud