Non-specific binding

Hi Everyone,

I've been having difficulty measuring the binding affinity of a small molecule analyte to an ~20 kDa protein immobilized onto a streptavidin chip. Because the same small molecule binds to another completely unrelated protein with similar kinetics/affinity, we believe this might mean we have non-specific binding. Our original buffer was 1xHBS-P+ 5%DMSO, but we've tried altering our detergent's (Polysorbate 20, i.e. Tween) concentration and increasing salt concentration and neither have gotten rid of non-specific binding.

The binding on-rate and off-rate appear very fast (approaching the instruments ability to fit) and when I try and analyze steady-state affinity data, I can't go high enough in concentration to get the response to level off, even at 0.5 mM (something we thought might also be indicative of non-specific binding). We are going to try altering our run buffer's pH next but we thought we'd see what others here thought.

Does this sound/look like non-specific binding to you all (see attached images)? If so, any suggestions on how to get rid of non-specific binding of this compound?

Thanks all,
J

It looks that the response proportional increases with the ligand concentration. If this are curves that are reference subtracted, I suspect non-specific binding or a bulk effect that is higher on the immobilized channel than on the reference.
You can look through the publications at for some extra information at www.sprpages.nl/experimental/non-specific

And in Giannetti, Anthony M. et al - Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors. Journal of Medicinal Chemistry 51: 574-580; (2008).A publication on how to identify and deal with promiscuous interactants. This article outlines a systematic approach to identify and classify promiscuous binders. In addition, it presents a number of datasets from poorly behaved to well behaved.

Kind regards
Arnoud

Thanks Arnoud for the fast response! The Gianetti et al. paper (Figure 3B) looks like what we're experiencing though I'm still trying to see what they say we can do to alleviate this problematic compound behavior. I have a couple of follow-up questions though.

1. What would cause a higher bulk response in the immobilized channel than on the reference channel? This doesn't happen with other analytes we look at with this same chip.
2. Do you know why we might be seeing binding of this analyte to other unrelated protein targets or have any personal suggestions on how we can alleviate non-specific binding?

Thanks again,
J

1. It is possible that due to differences between the reference and active channel there the excluded buffer volume is different. Especially with DMSO and glycerol this can give a 'bulk effect' after reference subtraction ( www.sprpages.nl/troubleshooting/bulk-and-spikes ). But is this is not the case with other analytes, the conclusion goes to non-specific binding to either the ligand or sensorchip matrix.

2. Integrity op a protein is a difficult thing. Some proteins and compounds can resist standing at 4 degfrees C for days, some don't like it for a short time. Freeze, thaw cycles can damage your protein/compound and also the formulation (buffer) has a strong effect on integrity (folding, aggregation, etc). You see, dificult to give an answer without exactly knowing all of your components and procedures. 

I hope this will give some idea's.
Arnoud

Thanks Arnoud for the response! Returning to your response to #2. We know that both the immobilized on-target and off-target proteins are stable at the conditions we’ve used them based off past SPR work and assays performed on these proteins. The off-target protein (where we shouldn’t expect analyte binding) is BCA.

In an attempt to remove non-specific compound binding of this analyte to off-target proteins we’ve tried the following buffers:
HBS-P (10 mM HEPES, 150 mM NaCl and 0.005% v/v Surfactant P20)
HBS-P+ (10 mM HEPES, 150 mM NaCl and 0.05% v/v Surfactant P20),
HBS-P+ 2xNaCl (10 mM HEPES, 300 mM NaCl and 0.05% v/v Surfactant P20)

We know that this analyte should interact with our on-target protein specifically based on evidence from the literature such as IC50/EC50 data. We also know that both proteins are stable in these buffers.

Do you have any other ideas on how we can improve this problematic analyte behavior?