How to optimize an experiment for a lower affinity analyte

Hi all,
I am new to SPR and have recently started using a BiaCore T200 and a neutravidin chip coated with monoclonals.
I assessed glycan binding to the lectin, SNA (Kd in the nanomolar range), which is pretty high for lectin interaction. The response units of the coated monoclonals I used in multiple experiments ranged from 60 to about 120. At these RU (ligand) values, I obtained nice looking SNA-binding data. Next however, I want to assess lectin interactions that are in the micromolar range. How should I modify my RU (ligand) to still obtain usable data for lectin interactions that have a 1000 times lower Kd? Any pointers or suggestions? Anything would be appreciated.

Thanks!

Hi,

You can use the same monoclonal surface density like the other lectin. However you need to use a higher concentration of analyte to get a comparable response like the strong binding lectin.
You can use the kinetic titration to get a quick indication of which analyte concentration you need ( www.sprpages.nl/experiments/strategy ).

kind regards
Arnoud

Thanks a lot for your response Arnoud! Can one assume that the effects of the affinity and concentration on the RU are inversely correlated? e.g. an analyte with a thousand times lower affinity for the ligand, requires a thousand times higher concentration of analyte to generate the same RU?

Yes,
Rememder the definition of the K_D: the analyte concentration necessary to saturate 50% of the ligand.
So with a K_D of 1 nM you need 1 nM and with a K_D of 1 uM you need 1 uM.

Arnoud

Thanks so much for the concise input!