Conditioning NTA chip and Ni not loading on chip

Hello,
I have a 2 part question. I am a newbie and I have been trying to optimize protein-protein interaction on the Biacore 3000. I want to use the NTA chip as one of my proteins is his tagged.
I ordered the NTA chip and conditioned it using the MDL method suggested (with somebody's help). Then, I used it 5-6 times. I only used cells 3 and 4. The Ni was loading properly but after some time (7th or 8th I believe) the Ni stopped loading. I did switch from HBSP buffer to DPBS (withou Mg or Ca) because P20 was interfering with protein interaction. Then all at once, none of the cells would load with Ni. Even the GE rep was at a loss to explain this phenomenon.

I ordered another chip and tried to condition and load Ni. However, I made a mistake and instead of using the recommended Method file, I manually injected Regeneration solution followed by 2 buffer washes. But after that step, there was no change after Ni injection. I am not sure what is going on. Tomorrow I will redo the conditioning (using the right method) and try to load the Ni. But I want to get some insight on what I could be doing wrong. Can someone please help.

1. What could have gone wrong with the first chip that it would stop responding to Ni Loading after a few runs (of ni loading, ligand binding and regeneration)
2. Did I damage the new chip irreparably by not using the recommended method and just injecting the regeneration solution.
Please help
Thanks in advance
H

Hi,

Do you use a single flow solution or two? Look at www.sprpages.nl/sensor-chips-intro/biacore-sensor-chips/nta for an explanation.

Since you do not state the regeneration solution I assume that u use the 350 mM EDTA to strip the Ni from the NTA. However, this is not always necessary since you only want to remove the ligand. You can use immidazole to strip the ligand from the Ni. This can extend the lifetime of the sensor chip.

Main question is, when you load the Ni, load the ligand and strip the sensor chip, what are the baseline responses? The baseline response before Ni loading should be comparable with the baseline after removeing the Ni in the last step. If your ligand is denaturing under regeneration conditions, you have a risk that you cover the surface with protein and be unable to regeneratie properly.

I don't think manual injections of the regeneration solution or via a method file will be any different for the surface.

What kind of reconditioning solutions do you use?

Kind regards

Arnoud