Forum :: Low protein signal during immobilisation


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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.

TOPIC: Low protein signal during immobilisation

Low protein signal during immobilisation 22 Aug 2014 14:56 #1

Hİ everybody,

I use Bionavi210A SPR. I'm still learning. I try to immobilze HSA with amino coupling on CMD (=CM5) chip. But my protein signal is very low. My coupling buffer is 10 mM Na Acetate pH: 5.2. HSA is globulin and fatty acid free. I tried a lot of different chemical such as different protein (BSA), different EDC and NHS, Different buffer ( 5 mM MES pH: 5.0) but nothing changes. I also tried different concentraion of proteins. I sent you my sensogram results.

HSA_2014-08-22-2.jpg


BSA_2014-08-22-2.jpg


I sent you my sensogram results for BSA and HSA .

Do you have any recommadation to solve this problem?

Low protein signal during immobilisation 23 Aug 2014 17:08 #2

Hi Biber,

Not sure what i see in your sensorgrams (immobilization or experiment; please explain) but i have some suggestions.
- take a fresh equilibrated sensor chip
- inject the HSA solution in different pH buffers (www.sprpages.nl/immobilization/ligand-pre-concentration.html)
- take the pH solution with the "best" pre concentration properties.
- then make fresh NHS and EDC according to the Biacore protocol or by the provided BioNavis protocol.
- Try again.
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