I did a study of the following buffers with the following NaCL concentrations - 0.15, 0.25, 0.35, 0.5M. 0.15M is what I typically use. Seems like after 0.15M it starts an inverted peak. Top one is 0.15, then 0.25, 0.35, and bottom is 0.5M. Does not make sense since back in 6-15, it worked...
I see you use automatic reference subtraction. How are the raw curves? 0.5 M should give you a huge upwards jump.
Now you find -50 RU (0.5 M NaCl) after reference subtraction. It could be that the two channels react differently to the salt concentration due to different surface immobilization. It seems to me this is the case here, because the effect is concentration dependend.
Changing the FC. hmmm, Is system check alright? Is buffer injection allright? (pipet directly out the buffer bottle)--> there should be no differences in channels and after reference subtraction.
I am still a bit confused about which curve you subtract from which curve. If you inject the 0.5 M NaCl solution do you record two curves (1= with ligand and 2 = reference)?
Looking at curve 6 and 7 I should say this are not raw data since the buffer jump should be different between 0.5 and 0.15 M. Do you change the running buffer also to 0.15 and 0.5 M?
Do you inject salt only (no analyte)? Then you should not expect a binding curve but a buffer jump and flat line. You can use this curve for compensating differences in channel responses (double referencing).
The other thing is that running in 0.5 M NaCl will diminish binding of a lot of proteins. In fact is is a know recipe to dissociate proteins from sensor chips or columns.
Sorry for the confusion. I did another simple experiment. Using FC 2, I injected the following: 1) 0.15M NaCL buffer only, 2) TSH in 0.15M NaCL, 3) 0.5M NaCL buffer only, and 4) TSH in 0.5M NaCL buffer. Each sample having each own cycle. I started with the 0.15M buffer and switched to the 0.5M buffer. The sensorgram below are labeled. Maybe try a diff. chip? running out of ideas...
That is funny, you should expect with 0.5 M the same as with 0.15 M. I am also out of options why this happens and how to solve.
Try another chip or an other ligand analyte pair to make sure the machine is working properly.