Fellows,
I'm looking for a simple protocol for immobilization of MP-11 as a ligand, on the Surface Plasmon Resonance gold chips.
I couldn't find anything in the literature and my graduate degree is not in chemistry! I'd highly appreciate the help if anyone knows of a specific protocol or even a close one that works on peroxidases.
MP-11 has a His tag by the way. Also I should mention that the device I'm working with is BI instruments 2000.
Looking forward to your replies. Thank you so very much.
What kind of sensor chips do you have available? Considering that MP-11 is a short peptide, I would use the His-tag for immobilization. You can either immobilize an anti-His antibody by standard amine coupling chemistry (I can send you the protocol if you want), and then perform an antibody-capture of MP-11, or you can use the rather expensive NTA coated sensor chips. With the NTA chip you do not need an antibody but in my projects, the NTA sensor chip has never worked really well.
you can refer one of our article and check the stability before you select the polymeric material for immobilisation .
A common approach is to use EDC/NHS to couple proteins or molecules to the surface. This requires that you first modify your gold with some sort of thiolated molecule that has a end terminal with, usually, a carboxyl group.
Since you mention that MP-11 has a His-tag some sort of NTA terminated self assembled monolayer might also work.
What kind of sensor chips do you have available? Considering that MP-11 is a short peptide, I would use the His-tag for immobilization. You can either immobilize an anti-His antibody by standard amine coupling chemistry (I can send you the protocol if you want), and then perform an antibody-capture of MP-11, or you can use the rather expensive NTA coated sensor chips. With the NTA chip you do not need an antibody but in my projects, the NTA sensor chip has never worked really well.
Christian,
Thank you so much for your great response. I would highly appreciate you sending me the His tag protocol if you don't mind. I have just the bare gold chips that need to be prepared. and this is my first time with immobilization and nothing has answered for the past 8 months .
I really appreciate you taking the time to read the question and answering it.
Please let me know of your further thoughts.
some His-tag proteins are more stable on the NTA chip and some His-tag proteins are not stable ( means base line is not stable after injection of His-tag protein) unable to start kinetics experiment. Need sugggestions .
Thank you very much
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