This is a very specific question.
In label-free assays like those used in Biacore, one often uses a carboxymethylated chip surface which tends to have a strong net negative charge. In the SPR binding curves, I often see that there is a residual non-specific binding signal, especially in sera and other messy media. These interactions can usually be washed off in a higher ph solution.
We are getting these types of responses; ie non-specific binding that can be eluted in ph 10. Our question is: should we work on making a more charge-neutral surface that is less likely to show this type of binding? Or since it can be washed off, are we better off to just perform elutions after all of our experiments and assume the residual bound ligand is bound specifically?
I was surprised to see how prevalent these non-specific effects are, and it seems like most sensors just rinse off any resdiuals and don't worry about these attractions.
PS, we are in the process of trying to make our sensor more charge-neutral, exploring various means of blocking, quenching, P20 detergent use and reducing the carboxylation level on the surface. My question is more in regard to how common the practice of just ignoring these effects is.
Could you be more specific about your assay? Do you graft DNA, an antibody, enzyme... to the surface?
Non-specific binding is quite normal when analizing complex mixtures. I guess you might have considered it already but, are you blocking the surface? (e.g. BSA, casein, PEG).
At last, in general if you increase washing stringency you should expect to remove non-specifically bound molecules before those otherwise specifically bound (again, to what?). However, the maximum detectable signals decrease and may compromise your limits of detection (because if you set the protocol with a harsh eluting condition, then you should stick to it for all subsequent experiments).
Sorry that I had missed your answers, guys, This is a fairly old question, and we were able to reduce the non-specific binding through the use of tween detergent in our running buffer. It does seem to be a purely electrostatic process; however, it's also found in biacore sensors and other label-free sensors, just because to create the surface, one has to concentrate a lot of charged things in a small area. This makes a charge-sink that would not occur in a real organism (ie in the bloodstream), and it can be screened with salt/ph etc...
If you are using amine coupling as your immobilization strategy, you can block the dextran sensor surface with ethylened diamine instead of ethanolamine to help neutralize the negative charge on the surface at pH 7.4. In addition, as you mentioned detergent helps a lot with non-specific binding but you can also add some free dextran in your running buffer to reduce it further.
@Phillip Page is there a recommended molecular weight for dextran as an additive into running buffer? They come in different sizes CMD-5, CMD-500? Do you think CMD as an additive will decrease non-specific binding in a MUA-11 (mercaptoundecanoic acid) SAM (self assembled monolayer) modified chip?
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