No binding with the protein

Hello everyone. 
I'm working on CM5 chip and now have troubles with protein binding. The different concentrations of protein diluted in the running buffer, HBS-EP+. Tried for several times to change something in the protocol, but it doesn't work, there is no binding between Aptamer and protein. 
Do you have any suggestions that can help? 

Will be very thankful for your advices. 

Appreciated,
Zhazira

Hi,

I assume immobilisation has worked as you expected and is stable? Do you have any more details of this, or of what the traces look like/concentrations, etc?

 

Hi!
Thank you for your reply,
Yes, immobilization works. Since I am using a CM5 chip, the activation step, immobilization of SA and Aptamer goes well. However, when I would like to see protein binding, the injections go well, but at the end there is no binding to the aptamer, the RU goes below the initial level, and stabilizes during the washing step. 

Also, concentrations are high, 2.5, 5, 10 micrograms/ml

Hi,

Are you able to give any other details of the ligand and analyte, like size, tags and such?

Some things to consider are the quality/stability of ligand and analyte. Certain tags/conjugations may also affect the binding site depending on the binding epitope, so it is worth considering this too. If it is commercially bought, it is worth checking the sequence and that the length of protein is correct (i.e. if it was a receptor binding a protein/antibody extracellularly, that the sequence for the protein covers the full ECD).

Another thing to check is that there is minimal binding to the reference flow cell, it could be that non-specific binding is causing it to normalise and not show binding. It is also worth checking that there is stability and no issue after regeneration between cycles, it could be that the ligand is damaged or falling off (although unlikely). 

Do you know that these two molecules actually do interact and bind? Is there already existing literature that state they do? 

Thanks

 

I have added some informative publications about aptamers in the literature section:
www.sprpages.nl/sensor-chips-menu/aptamers

Arnoud