my covalent immobilized protein is a receptor. For some ligands I am observing concentration dependent negative signals, the lowest concentration resulted in the most negative signal. After a specific concentration the signals turn into positive signals. I am observing this negative signals before referencing and after double referencing and I have no idea how to deal with this results.
Has anyone seen some similar results before?
Is the negative signal high (> 50 RU) or low?
Is the buffer of your analyte matching the running buffer?
Your are talking about some ligands, so not all of the channels have this fenomenon?
What happens if you inject running buffer only? When subtracting reference is the result near zero?
the negative signal is concentration dependent from -3 up to 20 RU (see above:)) and the buffers are matching each other, because I use the same buffers for running buffer and dilution series. In this case without DMSO.
I am sorry, I didn´t mean ligand, I meant the analytes and that I couldn´t find this phenomenom for all analytes.
I inject running buffer routinely in all my runs. For this run I have 12 RU for the ligand channel and 10 RU for the reference channel.
The results above are double referenced (reference channel and blank substracted)
I think you have a reference surface which is behaving a little different to the analyte injection than your ligand channel. Because of the low signals this is extra clear in your experiments. The difference is with some analyte you say and thats why you cannot compensate this with buffer injections.
You mention DMSO. Is your analyte in DMSO? Traces of DMSO difference can cause this. To compensate for DMSO difference you can make a calibration plot for the responses.