I ran two analytes in equal concentration of 1 uM through a surface with the ligand (upper graph) and a reference surface (lower graph). I am confused with the fact that during the injection of the first analyte (light blue, channel A2) through the surface with ligand the signal is decreasing. I saw the same effect in a separate experiment with lower concentration of the first analyte, so I don't think that it's an artifact. Have you had a similar experience with a signal decreasing during injection? What may be the cause?
The graphs on the picture are only double referenced, channel reference hasn't been applied due to nonspecific binding of the analytes to the reference channel.
Instrument: Bio-Rad Proteon
Running buffer: PBS (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl)
Ligand: protein, 24 kDa. Immobilization level: 4000 RU.
Analytes: proteins with MWs of 14.4 and 16.9 kDa
PBS was run through the channels A1, A4, A5, A6. A6 was used for double referencing.
Indeed this is puzzling.
It does not look like it is something with the buffers because your L3A2 surface does not give a negative curve.
Was the negative signal with the lower concentration less? Probably, so it can be analyte concentration dependent.
It looks like the negative signal is permanent, or is the curve after washing back to the baseline before the experiment?
Is your analyte enzymatic active? Probably not but did you check is on a gel for additional (contaminating) compounds?
Note on the responses: you have 4000 RU of a 24 kDa ligand and a 16.9 kDa analyte. Is the response in L2 not very low?
Sorry that I don't have a direct answer to your question.
Hope to hear from you when you find out what is going on.
this is not too unfamiliar as i have used the proteon several times before.
you may have to check your referencing, it is quite complicated with proteon.
since they use inter spot reference etc, like you said when there is non-specific binding to your reference channel, there may be some of these non-specific bindings on some of the interspots as well. so when there is more material binding onto the interspot, less to the ligand surface, when subtracted the curve will go downwards negative upon referencing
the proteon works like a breeze if interactions are simple and there is absolutely no interaction with the alginate matrix nor reference channels.
beyond that it is a bit more challenging. good luck!