Single cycle kinetics

hi,
I have been trying Single cycle kinetics in a capture method. need to know if i should immobilize both the active and the reference flow cell?

In principle, single cycle kinetics (kinetic titration) and multi cycle kinetics (MCK) do not differ in that a proper reference channel should be used to compensate for non-specific interaction and bulk refractive index differences. The best practise is to immobilize the capture molecule on the active and reference channel. If possible, capture a non related / mock ligand to the reference and the active ligand to the active channel. Otherwise leave the reference channel as is with the capture molecule.
When using MCK, normally zero analyte injections are used for double referencing. When using SCK you have to do a full run without analyte to have a curve to do the double referencing.

Kind regards
Arnoud

Thank you Arnoud. I am also facing problem in performing the kinetic assay for a very high affinity molecule. I am using a CM5 chip and i have tried both direct and capture method. The Kd is a problem as it goes out of my instrument specifications and hence i get a cross mark for my QC Tabs. So I also tried with an extended dissociation time, SCK, MCK and nothing is working . It would be of great help if you can recommend something to try.

It is better to post a new question in a separate post for clarity.
I come up with a longer dissociation time using the highest possible analyte concentration for maximal decay in the signal. You have to run the proper blanks with it to perform double referencing. In addition you can try to use a low ligand density to avoid rebinding.

Or you have to use other methods like the one in the publication by Drake.

1. Drake, A. W., Myszka, D. G. and Klakamp, S. L.; Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods. Analytical Biochemistry (328) 1: 35-43; 2004.
2. Katayama, M., Sato, T. and Kuromitsu, J.; Capture molecules preconditioned for kinetic analysis of high-affinity antigen antibody complex in Biacore A100. Analytical Biochemistry (424) 2: 168-177; 2012.
3. Katsamba, P. S., Navratilova, I., Calderon-Cacia, M., et al.; Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users. Analytical Biochemistry (352) 2: 208-221; 2006.

Arnoud