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?
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: