Immobilization of biotinylated protein on SA chip.

Hi,
I have tried immobilization of biotinylated protein on SA chip. I have manage to get only 700 to 800 RU. Biotinylation of protein was confirm by two different method ( western blot and quantification by kit ). Please suggest , how to improve the immobilization level up to 3000 RU.
Thank You,
Yogesh

Hi,

For the SA-sensor chip it is necessary that your biotinylated ligand is absolute free of free biotin since this will block the SA on the chip. In addition it is better to have 0.5-1 biotins per protein than many. In this way more protein can be bound to the surface.
Since you not state the size of the biotinylated ligand it is guessing how much 700 RU is in the amount of actual immobilized ligand (pmol/mm²).

Ligand_sites = R_ligand/Mr_ligand (pmol/mm²)

I tried to look up a reference on this but could not find it for now.

In addition if the size (in Da) of the analyte is in the order or larger compared to the ligand, 700 RU could be ok.

kind regards
Arnoud

Hi Yogesh,
we have observed huge differences in the capacity of SA-chips from different providers, varying by a factor of ten or more. It might be worthwhile checking out others than only the instrument manufacturer (if that is your source for chips).

Best regards,
Matthias