i am trying to find KD of aptamer as the analyte and protein. I'm using a CM sensor chip. I'm using PBS with tween 0.005% and 1 mM MgCl2 as RB. Normally I don't use ethanolamine to block the channel because aptamers don't have primary amine that can bind to the activated COO- and also we observed good binding without EA. we thought EA blocks everything and so we didn't use EA. with EA we couldn't generate any results. recently we got a good KD and it was pMol range. so it is too good to be true for this aptamer. when we checking specific binding, we realized there was some mistake in this experiment. so we activated the reference channel and injected the aptamer. we saw the binding, but that is not a non-specific binding. does someone have an idea about yhis? if that please help with this.
we assumed there should be a kind of positive charge interaction and that should be some kind of contaminants of the CM chip. it is a new CM chip. does anyone know any cleaning procedure for CM chip?
Protein over aptamer. protein is the ligand and aptamers is the analyte
Hello, your explanation is not so clear for me. But I will try to give you more info.
Your surface is negatively charged (carboxymethyl dextran) and your aptamer is also negatively charge. You have somehow a electrostatic repulsion and you are not seeing what you have to see.
-As I was working with aptamers, I suggest you to generate a biotinylated version of your aptamer and to immobilize it on FC2 on SA sensorchip. FC1 will be deactivated(same step than FC2 but no ligand injection).
Then, you inject your protein as analyte. The expected kD for protein/aptamer is pM-nM range.
Other things you could do are:
- calculate the theoretical Rmax expected to see. Compare you Rmax and the theoretical Rmax. If you have a significative difference, it means there is no real binding.
-how much of protein did you immobilized? If you immobilize too much, you can generate an avidity effect and then your KD is surestimated.
And I recommend you to block the reactive coo with ethanolamine as the standard procedure described in the sheet data. And FC1 and FC2 has to be immobilize in the same way exluding the ligand for FC1.
Hope it helps!
Best
Vanessa
Vanessa,
We generated SA chip by ourselves and successfully immobilize biotin-aptamer on the chip, but still, we can't observe the binding for the target protein.
This is very strange, so we did several control experiments: 1) we successfully observed the interaction between biotin-ssDNA and its cDNA; 2) we confirmed that the aptamer can bind the protein by flow cytometry and fluorescent microscope.
Could you pls give some advice?
Thanks.
XT
Dear Xiaohong Tan ,
Thank you for the updates. From your controls, I have several questions and suggestions.
- If you see an interaction between a ssDNA and its cDNA, it is good but it doesn"t mean that your aptamer is properly folded and functional. Did you do the step of " Re-folding process with heating and cool down) prior to inject your protein?
-Regarding the cellular assay, are you working with the same recombinant protein or it is expressed at the surface of the cell? I have some doubt now about the protein integrity. If Siddhartha followed my recommandations and no binding is observed, it could be that the protein is not functional or aggregated. It will be great to check the protein integrity (DSF, SEC-MALS).
-The last thing is: The binding site of the protein is closed to the NH2 biotin part of the aptamer. It cannot bind because it is not accessible. it will worth to test a Cterm biotin aptamer (if possible) to have a free N term. If it is not possible to try it, I will suggest you to try another technology with the 2 partners in solutions (MST, Nanotemper) easy to order a FAM-aptamer in C and N terminus)
Hope it helps,
Vanessa
Hi Vanessa,
Thank you again for your professional and kind help.
We use 5' FAM labelled aptamer to bind the protein, and verify good binding by flow cytometry and fluorescent imaging, so I believe the aptamer and protein for this SPR test are fine.
It is a good idea to try 3' biotinylation, and actually today I did order this one. Will let you know whether it work or not. It is very strange that we just can't use this BI 4500 for SPR analysis.
Thanks.
Best,
Shawn
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