Reference surface

Hi everybody,

I have a doubt. I have biotynilated antibody that I will capture over a streptavidin chip. I dont have any other biotnylated antibody that I could use for the reference spot. What would you recommend in this case? a bare Streptavidin spot? Activate and deactivate the chip? Covalenty immobilize an antibody on the surface?

Regards,
joseantonio

Hi,

I am using now a streptavidin chip and use as reference just the native surface with streptavidin on it. There is no background binding so this is ok.

Activating / deactivating is not usefull because you don't do that with the other surface. Actually it is immobilized with streptavidin. Probably you will introduce some modifiaction you do not want with an extra activation.
It is maybe better to saturate the reference surface with biotin.

Arnoud

I just saw what you mentioned, Arnout. On an activated-deactivated spot I get more background than in SAv-coated spot. I have D-biotin (Sigma) for blocking the SAv, does anyone know what concentration should I use?

Regards,
joseantonio

Start using 10 ug/ml and sature the surface. The response will be rather low (D-biotin is very smaal). Wait until it level out and wash thorougly befor switching to the other channels.

regards
Arnoud

Did it worked? If not, putting BSA (0,1 mg/ml) into the running buffer might help.
eya