Hi, I am trying to immobilize biotinylated protein to SA chip using biacore T200. The immobilization levels we are getting are low (800-1500RU). Our aim is to immobilize protein upto 4000-5000 RU. We have tried both ways of immobilization (time/flow rate and Aim of immobilization). Buffer strent for immobilization is ~15-20 mM of sodium phosphate. Protein concentration used is 50µg/ml -200µg/ml. Kindly suggest your opinion.
Thank you and best regards
Dear Gautam,
of course, you can try to optimize the immobilization conditions in order to increase the immobilized levels. For that, you can play with temperature and, more importantly on flow rate and time of contact. And if you already play with those parameters, it might be difficult to get higher immobilization levels.
But now, the point is: why do you want to immobilize such a high level of proteins?
Usually, higher the immobilization level, worst the quality of the detected interaction. You may encounter plenty of artifacts due to the high level, like: 1) parking problem or masking of potential ligand binding sites by previously formed complexes; 2) heterogeneity of coupled ligand; 3) rebinding of dissociated analyte; 4) mass transport limitation that occurs when transportation kinetics is slower than binding kinetics; or 5) nonspecific binding between ligand and analyte, ...
Definitely, the higher level is not the best for high quality SPR experiments.
Good luck for your experiments
Yves
Dear Yves,
Thank you for your input!
Since our analyte is a small molecule we wood like to have ~4000RU of immobilization to start with. I agree on your comments on the higher immobilization.
Thank you and best Regards
Gautam
Dear Gautam,
As an alternative, you may select to use a low level of immobilization, and run your small molecule in competitive mode using Label-Enhanced SPR to boost the sensitivity. Check out this paper:
https://doi.org/10.1016/j.ab.2016.06.008
Best,
Anders
Hi,
You can play with flow rate. You should keep low flow rate that allow the ligand come in conatct with SA.
Good Luck
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