we have encountered the following problem: After chip immobilization, up to 3 runs measuring antibody-FcyR binding could be performed, which resulted in correct sensorgrams without any problems. After the fourth run, however, the sensorgrams corresponding to the two replicates of the sample did not align and even showed a downward shift at the highest applied concentrations (see attached figures). I think we prepared three new CM5 chips, and this happened every time. We did not change the exerimental setup, we checked the capture levels and performed a system check, and everything was fine. Did anyone have the same problem or does anyone have any idea why this is happening and how to prevent it?
What kind of ligand and analyte? Please state the running buffer and regeneration condition if used.
The replicates, are they diluted every time new or standing the whole procedure in the instrument?
we measured the interaction between an antibody (analyte) and Fc gamma receptor (ligand), using 1X HBS-EP+ buffer as running buffer and 10 mM glycine-HCl at pH 1.5 for regeneration. The replicates were kept in the instrument during the entire run. These experimental conditions remained the same for all runs, however, after the third or fourth run, the problem occurs and we don't know if something is wrong with the chip or the instrument.
Since the first three runs work ok, I think you can rule out an instrument problem.
If I understand correctly, you put four replicate dilution series (five concentrations each) in the instrument and run the replicates. After the third it goes wrong.
What happens I you make each replicate just before the run starts? Do you have the same problem after the third run? No, this could indicate that something is happening in the vial during storage in the instrument.
Second, why do you use the regeneration since the dissociation seems quickly enough? Just wait some extra time. This will preserve the Fc gamma receptor better (Glycine pH 1.5 seems a little harsh and we prefer 0.85% phosphoric acid in these cases).
Third, what is striking on the sensorgrams is that they go below zero. Therefore, check your reference channel because I could be that there is something wrong with non-specific binding.