Immobilization?

Hello,

What do you think about immobilization of a trimeric protein on the sensor chip CM5? . I am concerned about trimeric form of the protein after regeneration.

Isoelectric point of the protein is 9,63. So it is not good candidate for analyte in spr. And indeed, initial experiments (Biacore 3000; Running buffer: 50 mM TRIS pH 7.4, 150 mM NaCl, 0.005% P20 , 0.02% NaN3, 0.1 mg/ml BSA) indicated non-specific binding.

Thank you.

Dorothy

Hi Dorothy,

This is always a difficult one. Even if your protein is monomeric because regeneration can alter the tertair structure.
The protein will probably bennefit from the amine coupling by coupling the protein at multiple points and thus make is more stable on the chip.
If you have a positive binding control you can try different regenerations and observe if this interaction is stable.
Try the regeneration method explained at www.sprpages.nl/kinetics/regeneration.html
This will help you to select the mildest regeneration condition.

You can try size exclusion chromotograpy under regeneration conditions to see if the protein is still trimeric under these conditions.

Arnoud

Thank you a lot.

Dorothy