Biacore T200, Immobilization stability

Hi,

I'm SPR user and I recently used biacore T200

I use the Biacore T200 wizard to immobilize the chip. In my experience, however, I have found that the amount of immobilized ligand is very irregular.

When I tried to trace the cause of this phenomenon, I was able to confirm that there is always a difference of about +/-50RU (I aimed to immobilze my ligand about 800 RU)

This phenomenon was exacerbated by immobilization immediately after desorbing, confirming that only about half of the ligands were immobilized (about 400RU).

Due to these problems, it is difficult to perform a stable analysis of binding kinetics like KD.

So if you have any idea or solution about this problem, I really appreciate if you tell me any advice.

Thanks for reading!

Hi,
The immobilization wizard is an algorithm that tries to get the immobilization level you ask for. However it has no clue about the immobilization as such. So first it checks the pre-concentration speed to determine it is high enough to reach the asked level and low enough to control the immobilization speed to avoid overshoot. This means that there is a matrix of best pre-concentration speeds (ligand concentrations) and immobilization levels. Although the wizard gives you the feeling that there it is a 'one size fits all' solution it is not and can benefit from some optimization. Immobilizations with a lower pre-concentration speeds seems to be more accurate.
Most users don't bother and accept an immobilization within a range of the asked immobilization level.

Desorb can have an effect on the ligand immobilization and subsequent analyte binding. Desorb is designed to clean the system tubing and injection system of adsorbed proteins. Thus when cleaning the tubing the system is left with surfaces that can bind proteins. In your case it seems that your ligand is easily adsorbed to the tubing although this should not have an effect on the immobilization level. This is because the adsorbed ligand is not immobilized and thus not detected for the immobilization level.
I would run the system for some hours on flow buffer before attempt an immobilization instead of immobilizing directly after a desorb. We normally desorb and sanitize at the end of a day and leave the system overnight for washing.

Due to these problems, it is difficult to perform a stable analysis of binding kinetics like KD.

The immobilization level should not have any influence on the kinetics and the KD. Thus -/+ 50 RU on ligand immobilization should give you the same answers.

Kind regards
Arnoud

Dear Aroud,

Thank you very much for your kind advices.

I realized that I made some mistakes.

One of my mistake is that the experiment was performed without enough time after desorbing.

Also, I used too high concentration of Ligand before.

I will try to experiment according to your advice.

Thanks to your advices, I get lots of new knowledge.

Have a nice day!