First, Shawn, what is your instrument supplier's opinion? Seems that there is none?!

From my perspective, it looks like an immobilisation effect (because you see a binding in flow cytometry or fluorescent imaging). Hopefully, the binding aptamer is big enough to get detected by SPR? If yes, either the immob protein's binding pocket ist blocked so that the added aptamer cannot bind - or the immob aptamer is sterically hindered to bind to the added protein.

Not sure what to do except to try different immob strategies, e.g., to try a long (6 C) linker between biotin and the species to be immobiized. Good luck!

Yours,

Hans Trutnau

Inventor of multi-step kinetics (aka single-cycle kinetics or kinetic titration)

Hi Hans,

Thanks for your respsonse.

I try to solve this problem for the past several months and the instrument supplier can't help too much for this question. To overcome the negative charges of dextran, we finally decided to use biotin/avidin interaction to immobilize the DNA aptamer. Although we were able to observed good binding using other methods, we still can't see any SPR binding. I did use a 10 T bases as the space between biotin and aptamer, but it didn't help.

Very strange! I know using SPR to study aptamer/protein interaction is very common but we just can't use this BI4500 to realize the goal, no matter what immobilization methods, such as dextran, NTA-Ni or avidin chips, used.

Thanks for your reply, Shawn. It's good that you tried the 10 T bases spacer; but it's indeed very strange that it didn't work. Did you try to link the biotin/spacer at a different aptamer position?

I would stress the instrument supplier's application lab to get a solution; they make money by selling instruments - and they have to support the customers!

Yours,

Hans

Thank you Hans. I ordered a biotin at the 3' and will try it again. I did try to get help from Biosensing Instrument but they can't really help us. Probably it's time to try other SPR instruments.

Thanks.

Shawn

Good luck, Shawn!

In case you have success, I would like to hear from you.

Yours,

Hans