Hello Mengxi,

For an aptamer binding studies, I will recommand you to have a biotin version of aptamer that can be captured over a SA sensorchip. Then, the protein is injected.

If the quantity of protein is a limited factor and you need to inject the aptamer, you can add some additives in you analyte sample ( NSB reducer cytiva :1X up to 5X If needed). You can optimize the buffer ( Higher salt concentration 500 mM nacl, decrease the pH ..)

Hope it helps...

Vanessa

Hi Mengxi Zheng,

In case of the His-tagged protein captured on the NTA-surfase, it may well be that the His-tag blocks/hides the protein's binding site, which also may be a reason for your negative results.

Try the ligand/analyte pair the other way round and immobilze the biotinylated aptamer on an avidin chip (as recommended before by Vanessa Porkolab), and, in case the aptamer binds in a deep protein pocket, insert a rather long spacer between biotin and aptamer (min C6) in order to achive a good accessibility of the aptamer.

And since the biotin/avidin capture binding is very strong, you may also try then a multi-step kinetics run (MSK as originally invented by me, aka kinetic titration or single-cycle kinetics, SCK, as called by Biacore users - in fact, MSK is exactly the same as SCK: multiple consecutive steps of different analyte concentrations and intersected buffer flows in a single run without the need of intermediate regenerations). Please take care that the MSK/SCK methodology is covered by patents that were taken over by Biacore (now GE Healthcare) from me in 2006 and is therefore restricted to Biacore users.

Good luck and, please, reply then!

Hans

I agree with Vanessa Porkolab.