Fitting does not work and drop in response

Hi,
I've recently measured protein-DNA interactions on a streptavidin-coated chip, where biotinylated DNA was immobilized and an 8 kDa protein was used as an analyte on a Reichert SR7000DC. Unfortunately, it is not possible to fit the obtained curves, even if the highest concentration of 500 nM is excluded, due to the drop in signal. The drop in signal was only observed during protein injection, not during salt injections under the same conditions. I already checked analyte and ligand for purity and tested different flow rates, but the signal is mainly the same.

Also, it is not possible to fit the dissociation separately via TraceDrawer software to use the value for further fits of the association.
Does anyone have any idea what the problem could be and how to solve it?

I would greatly appreciate any help you can give!

Hi,
One thing I noticed is that the start of the association has a little 'hollow' curve and then goes faster upward. Since the buffer injection was ok, it can be something intrinsic of the interaction between the protein and DNA. The signal seems high enough, thus lowering the biotinylated-DNA concentration on the chip can lower the effect and maybe solve the 'dip' problem in the highest concentration used. Since the injection time is long enough you can try to do a steady state affinity analysis (without the 500 nM) to get at least an overall indication of the K_D. To try a different program to fit the dissociation you can look at Anabel ( anabel.skscience.org/anabel-1/?2490307#quick_start ). It is easy to use but first read the manual because it is not intuitive at first glance.
Kind regards
Arnoud

Dear Arnoud,

thank you very much for your advice! I will try it with Anabel!

Kind regards
Anna