high Rmax

Hi all!
I was wondering if it is possible to have an Rmax of 2000 RU on a BiaT200...
I know that the correct range is from 20 to 100RU but if I capture too much ligand, can I obtain a response over 2000RU or it is impossible and I will see and Rmax lower than expected?
Thank you all in advance
Nad

HI,
A theoretical Rmax of 2000 RU must be possible on the T200. If you reach the 2000 RU depends on the ligand - analyte size ratio, the amount of bound ligand, the kinetics and the analyte concentration. Most possibly you will not reach the 2000 RU but you don't need to to have kinetics.
Main concern with a high RMax is mass transport limitation and non-linear kinetics.
As recommended a lower Rmax is better (for kinetics)

Kind regards
Arnoud

Hi,
Really thank you for kindly reply.
In your opinion, for a ligand of 150kDa and an analyte of 333kDa and a capture level of about 2000RU, what could be the reason why my Rmax is really lower (100fold) than expected?
Thanks in advance
Nadine

Hi Nadine,
For a 2000 RU immobilization of a 150 kDa ligand a 333 kDa analyte has a theoretical Rmax of 4440 RU. Depending on the immobilization technique used, not all ligand molecules are still biologically active. For instance, covalent coupling chemistries can provide highly active biological surfaces, but when the reactive group used for the covalent binding is close to the interaction site, the immobilized ligand activity can be severely diminished. I can also think of crowding by the larger analyte molecule which could shield more than one ligand per binding.
You can try to immobilize less to see of the ratio between immobilization and analyte binding improves.
If possible immobilize a non-blocking antibody against the ligand, capture the ligand and try to bind the analyte.

Arnoud

Hi Arnoud,
Ok, perfect! Really thank you for your kind replay!
Have a nice day
Best
Nadine