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TOPIC: Need help optimizing NTA chip SPR

Need help optimizing NTA chip SPR 24 Jun 2020 06:05 #7

  • peterboy
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Thank you for all your help so far Arnoud, I've implemented everything you've suggested... Yet still haven't been able to get good SPR results for my Protein (ligand) - DNA (analyte) experiment on NTA chip.

I've attached detailed data graphs that I got from the Biacore machine. These are sensograms for Fc = 2-1, Fc 1 and Fc 2. Also included the Kinetic/Affinity curves for my experiments. I used all buffers suggested in the Biacore NTA chip page.

As you can see I keep getting the large peaks at the beginning and end of sample contact times. What could be happening? Has anyone experienced this before?
My sample analyte is DNA and they are in stock H2O, then diluted into running buffer (10 mM HEPES, 150 mM NaCl, 50 µM EDTA. 0.005% Surfactant P20, pH 7.4).
My guess is that this running buffer doesn't work well with DNA analyte? Could another reason being is that my protein ligand is stored in stock 30% glycerol and I should buffer exchange it with running buffer beforehand?

Another guess is that I should buffer exchange my glycerol stored protein into my senior's buffer (25mM Tris-HCl pH 7.5, 100mM NaCl, 20mM imidazole, 0.005% Tween20, 0.5mg/ml BSA)?

Any suggestion would be more than welcome, as I have been trying to optimizing this experiment for over a month now.
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Need help optimizing NTA chip SPR 24 Jun 2020 19:46 #8

Next time, please remove the regeneration, that will make the capture and analyte injection more visible.
In addition, transfer the curves to Y=0 before the first injection, that makes comparing of the response more easy.
It is now impossible to see the responses of the DNA injection.

I suspect that the first green injection is the ligand capture. There is a lot of bulk effect (> 7000 RU?). In principle, if the ligand is binding not a real problem but dialysing to running buffer could give you a clearer picture of the capture.

The injection peaks are probably mainly from the buffer mismatch. It depends on the dilution  how much water you dilute in the running buffer. This will give a slight difference in salt content between diluted sample en running buffer, and will be different for each dilution. To avoid that, you could dialyse the DNA. DNA is quite robust, so I don't think that there is a buffer problem there.

Unfortunately it looks like that there are negative curves after blank subtraction. This could indicate that the reference is binding relative more DNA compared to the active channel or this is an artefact due to the buffer mismatch.
My suggestion would be to dialyse all compounds to one buffer either Biacore's or your senior's.

regards
Arnoud

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