Negative signal upon injection of running buffer

Hi.
I am pretty new to SPR, and I encountered an issue I do not know how to deal with; and I am hoping that someone with more knowledge of the system can help me.

In a buffer system (HBS pH= 7.4, with 2mM Ca and 0.006% GDN detergent) I get negative signals even if I inject the running buffer. I am using the SAD200M chip from Xantec on Biocore T200.

Basically I equilibrate the system, wait until the signal is stable. Then I inject the exact same running buffer that the system is in (taken from the same bottle) and both the reference cell, and the cell where I immobilized my target membrane protein, show a negative peak. After the subtraction of the reference cell, the signal is still negative. This peak decreases continuously until the injection stops, then the signal returns promptly to the baseline. I observe this, until I completely overload the chip with the ligand (ca 1-15 µM of a Nanobody).

I don't know how to overcome this issue, and from the looks of it, double referenced method doesn't correct this issue. I did not observe such behavior with 0.05% DDM or 0.05% Tween-20 as detergent, but again the same issue occurred with 0.05% DDM and 0.005% CHS detergent mixture. Adding BSA does not help either, and since both cells are negative I would assume it is not unspecific adsorption.

Both CHS and GDN are cholesterol based detergents, but I doubt this would cause such an issue. I hope someone could give me a tip. Unfortunately, the proteins are unstable in buffers that have a different detergent.

Thank you in advance, Marton

As you write, the system can be equilibrated until a base line. When you do blank injections with buffer direct from the flow buffer bottle, do you still have this negative response? How big is the negative response?
If I have it correct the flow buffer with detergent is necessary to keep the ligand on the chip in the correct conformation. When diluting the nanobody, is the nanobody stock solution in the same buffer with detergent? Diluting the nanobody from a different solution can alter the injected buffer inducing negative curves. The use of DMSO in flow and sample is a classic example of this problem because small differences can give either positive or negative responses. You can try to compensate with an 'excluded volume correction' ( www.sprpages.nl/experiments/data-processing ) when the negative response is not to large.

Kind regards
Arnoud

Thank you very much for the answer.
The negative signal occures during blank injection aswell (injecting the running buffer) and is about 20 RU, and I dont know what causes it. It is literaöly the same buffer.
I dillute the nanobodies ca 1000 fold in the running buffer, and they do not contain any DMSO.
Yes, I always waot till the baseline is stable before I begin even with the blank injection.
The same issue didnt occurre when I had a different ligand and used different detergents. Nanobodies were in the same buffer before dillution, and blank injection didnt cause a negative signal.
Yes, the detergent is crucial for the ligands stability.

I attach a picture from a blank injection.