capture level really low

Hi everybody!
I'm quite new to SPR but I'm having quite a lot of issues with the capture level of my protein on the chip.
I'm trying to purify my protein from mammalian cells and perform the experiment using an CM5 chip.
I tried to do this with two different tags on my protein: GST-(2X)HIS-protein and (2X)HIS-FLAG-protein.
I've tried to isolate my protein either doing a GST-IP, a Ni-NTA IP (both followed by buffer exchange) or a FLAG-IP.
everytime the capture levels of my protein were too low (200-1000 RU Max) and we aimed at least at 5000RU.

Do you have any suggestions?
as running buffer I've been using 10 mM HEPES ph 7.5, 150 mM NaCl, 0.05% P20.
Thank you

You have to make your question a bit more clear. Are you trying to capture your protein on a CM5 chip with the tags or have you purified protein and do you use the amine coupling. The size of the protein to be immobilized determines largely what your capture level will be. In the case of capturing via a HIS or GST-tag the affinity is largly determining what your capture level can be.

Thank you for your reply. Sorry Iade a mistake on my original question. I'm using NTA chip for this experiment, that's why I have a 2xhis tag on the protein. I've tried to purify it either with ni-nta ip, flag ip or gst ip. However, in each of my experiments the capture level was really low...

Hi,
Maybe you can get some clues how to optimize the binding at www.sprpages.nl/sensor-chips-intro/biacore-sensor-chips/nta . I have also three publication that might be of help.
As an alternative you can immobilize an anti HIS-tag antibody and then capture the HIS-protein. This is probably much more stable.
Kind regards
Arnoud

1. Gershon, P. D. and Khilko, S.; Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector. Journal of Immunological Methods (183): 65-76; 1995.
2. Huang, Z., Hwang, P., Watson, D. S., et al.; Tris-Nitrilotriacetic Acids of Subnanomolar Affinity Toward Hexahistidine Tagged Molecules. Bioconjugate Chemistry (20): 1667-1672; 2009.
3. Nieba, L., Nieba, A. S. E., Persson, A., et al.; BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Analytical Biochemistry (252): 217-228; 1997.