Baseline increases between cycles

Hi all,

I am using the NTA sensor chip for my binding studies, but the baseline is increasing between each cycle.

At first, I thought it was a regeneration problem, so in addition to 350 mM EDTA, I have also tried 6 M urea and 0.5% SDS. The baseline is still around 1800 RU. I'm not sure what the problem is.

Briefly, my setup is:
Model: BiAcore X100
Sensor Chip: NTA
Ligand: 5 ug/ml of EGFR-his6
Analyte: Cetuximab or cetuximab-conjugated liposomes (haven't tested the liposome yet)
Running buffer: PBS

Will desorb/sanitize help (it's actually overdue)?

Thank you all for your help!

Hi Thiophylline,

Welcome to the SPRpages.

yes, please start with cleaning the instrument. And make new fresh solutions. That will give you the best starting point.

Now dock the sensorchip and regenerate with EDTA. This should give you the lowest baseline (NTI minus Ni).
Then load the Ni and observe if the baseline stays stable.
The load your EGFR-his6 and observe the baseline, then regenerate to establish if your regeneration is stripping all of your EGFR from the chip. Alternatively you can use 50 mM immidazole to strip off the EGFR. Then the Ni should be retained on the chip.
This procedure will establish if the problem is your ligand.
Then inject on an NTI-NI chip (no EGFR) the Cetuximab. It should not bind or only give a minor background response.
Also check how the surface behaves after the Cetuximab injection on the regeneration solution.
This will give you information about the analyte.

Let me know what you find.

Kind regards

Arnoud


To pinpoint the problem