SPR CAP-chip

Dear All,
good day. Hope you're doing well. I have some problems with blocking step on the CAP chip. Does anyone know how to solve problem: After the injecting blocking reagent, the RU increases and decreases until the baseline, wash away everything (SA+ligand).

Hi, and welcome.

Why do you need a blocking step? And what is you blocking solution?

kind regards
Arnoud

Thanks for your reply,
What about non-specific binding? Is it okay to skip this step?
I was wondering to have non-specific binding on the CAP chip surface.
Blocking solutions, ethanolamine and biotin.

Ethanol amine definitly not. This is a capture approach not an covalent immobilization.
Biotin? Maybe, when you are concerned about free avidin bindin sites, but if your analyte is free of biotin, I wont be bothered.
But, make a reference surface without your ligand to test non-specific interaction to the surface. This wil give you information if biotin blocking is necessary.

Arnoud

Hi,
Depending on which Biacore instrument you are using, you may not want to inject free biotin at all, even if you are seeing non-specific binding. If you have a serial flow cell system, the biotin can leak into other flow cells and obscure analysis/ interpretation. If you are having issues with non-specific binding to the CAP chip surface, I'd recommend removing biotin from the experiment altogether (if possible). For example, try capture with a his-tag or other tag that your ligand might have. I have seen NSB to streptavidin with many of the peptides I analyze. And as Arnoud suggested, use the reference surface for subtraction.