Forum :: Problems with capturing on NTA chip


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

TOPIC: Problems with capturing on NTA chip

Problems with capturing on NTA chip 12 Feb 2015 08:26 #1

Hello. I tried to capture 100 kDa 6His-tagged protein on sensor chip HTG (BioRad chip with NTA) using concentration 25 mkg/ml, flow rate 25 mkl/min and time 300 sec. Buffer - PBS with 0,005% Tween 20. I expected to get 1000-2000 RU (I need this capturing level to study interaction with 6 kDa peptide). But I've got only 100 RU! Recently I tried to use amine coupling chemistry (and get higher immobilization level) but the protein is not active under amine coupling. What should I do?
Thanks,
Alexandra

Problems with capturing on NTA chip 12 Feb 2015 08:55 #2

Maybe anyone else knows better conditions for your capture approach but if not, I would suggest trying anti-His-antibodies (tethered to a standard CMD chip via amine coupling) instead of NTA. Maybe this will result in a stronger binding.

Here is a link to a document from GE including this procedure:

www.gelifesciences.com/gehcls_images/GEL...9_20120116104450.pdf
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