I'm having a lot of trouble with optimizing DNA (aptamer) to protein binding on an NTA chip. I'm a newbie at SPR so any advice would be much appreciated. My protein does have a his-tag on it.
1. Do the running buffer, ligand buffer and analyte buffer all have to match? Ie. the running buffer used in the startup cycles, the buffer used to dilute the ligand and the analytes to appropriate concentrations.
2. The running buffer I use is: 25mM Tris-HCl pH 7.5, 100mM NaCl, 20mM imidazole, 0.005% Tween20, 0.5mg/ml BSA. When I run a startup cycle, injection of this buffer results in a noticeable increase in RU. I have also tried buffer: 10 mM HEPES, 150 mM NaCl, 50 µM EDTA. 0.005% Surfactant P20, pH 7.4. But that also produces an RU increase in the startup cycle.
I have read that injection of the running buffer should not do this and it should be almost flat? How do I solve this issue?
3. Up until now I have matched my running buffer, analyte buffer and ligand buffer. The resulting Fc=2-1 I get is flat, with some minor sharp dips and peaks at points of analyte injection. Is this to be expected?
Basically, I'm trying to optimize so that the startup injection of buffer and the injection of analyte will not produce a noticeable RU jump, in order to get accurate results. Am I going about this the right way?
1) Yes, it will you give better and understandable results that are more easily processed.
2) I would let out the imidazole since this will compete for the HIS-tag binding on the NTA surface. If you inject the running buffer (pipetted directly from the bottle in the machine) it should give an almost flat line.
3) Yes for the peaks if the buffer is not matched. Keep in mind that most instruments have channels that are connected in series which leads to a small time delay between the channels. That will give some peaks when subtracted before properly aligning.
Thank you very much for your helpful suggestions Arnoud!
I'd like to ask a few more questions if I can.
1) My protein ligand is stored in 40% glycerol stock, would I need to buffer exchange to running buffer in order to successfully conduct the SPR experiment?
2) When making the regeneration and conditioning buffers, is diluted to 0.35M EDTA using the running buffer (25mM Tris-HCl pH 7.5, 100mM NaCl, 0.005% Tween20, 0.5mg/ml BSA) suitable?
3) My senior has recommended using the above running buffer (instead of 3mM EDTA) as the wash buffer, is that appropriate?
1) You can dilute from the DMSO but be aware that DMSO give a high bulk effect even at low (<1%) concentrations. I don't know if DMSO is compatible with HIS binding.
2) for regeneration 0.35 mM EDTA is OK but be aware that you also strip the Nickle from the NTA chip and you have to reload the NTA with nickle to work again.
3) I would not recommend because of the above. Stick to the low EDTA (0.05 -3 mM).
I forgot to mention that my experiment is looking at the binding between His-tagged protein (ligand) and DNA aptamer (analyte).
Because of that, would the running buffer need to be changed from (10 mM HEPES, 150 mM NaCl, 50 µM EDTA. 0.005% Surfactant P20, pH 7.4) to something else? Could you help suggest any possible running buffers?
My senior has published on such an experiment (involving a different His-tagged protein, but still with aptamer analyte) and successfully used the running buffer: 25mM Tris-HCl pH 7.5, 100mM NaCl, 20mM imidazole, 0.005% Tween20, 0.5mg/ml BSA.
This is the methodology quoted:
"Surface plasmon resonance (SPR) measurement was collected using a Biacore X100 instrument (GE Healthcare). PvLDH was captured on the surface of NTA sensor chip (GE Healthcare). A running buffer containing 25 mM Tris-HCl, pH7.5, 100 mM NaCl, 20 mM imidazole, 0.005% (v/v) Tween 20 and 0.5 mg/mL BSA was used for ligand capturing. The surface of NTA chip was primed with running buffer and PvLDH cubamer, 1501s, was injected in at a flow rate of 30 μL min−1 and at 25 °C. All data were referenced for surface without captured cubamer and blank injections of buffer. Sensorgrams were analyzed with Biacore X100 Plus Package evaluation software (GE Healthcare)."
However, I am unsure of including the imidazole and BSA. In addition, would it be wise to use this running buffer in all my solution preparations as baseline or just for ligand capturing?
If the buffer in the reference worked previously and your set-up is comparable, you should use that. When your His-tagged protein is not binding to the NTA chip, lower or leave out the imidazole. The BSA should not have a big effect. First have a proper ligand binding and go on from there.