2 replies to this topic

[PippiLa]

Hi there!

Does anyone have a good working protocol for the conjugation of peptide to a carrier protein? (Doesn't matter if BSA, Ova, KHL). I need it for ELISA plate coating.
I think that the glutaraldehyde method would be best, as it is said to couple at the N-term of the peptide. But I am thankful for any suggestions.
I know the very best is peptide synthesis with an additional Cys at the N-term, but, well, peptide is already there - no Cys, so I will have to work with what I've got
Is there a possibility of influencing or at least checking the coupling ratio of protein:peptide?

Thank you all for help and support!

[BioMiha]

If you're only looking to do an ELISA you're better off just coating the peptide on the plate. We do this all the time using your standard ELISA plates from Corning or Nunc. If you're worried that the peptide won't adsorb well (I have not come across one that hasn't but well I'm still young...) you can couple the peptide covalently to amine activated plates - Nunc and Pierce sell them.
If you really want to couple the peptide to a carrier protein, just a word of warning. If you use a bidirectional linker like glutaraldehyde or EDC it is very likely that the cross-conjugate polymers will not be soluble. At least that is my experience. It all turned into a big mess.

[PippiLa]

Thanks a lot! Yes it's about ELISA design.
For me that's a completely new idea of coupling the peptide directly to the ELISA plate. I have done a lot ELISAs already, but at least in our group it was always thought that the antibody would not be able to bind the peptide, which is directly bound to the plate, simply because there is not enough space.
So the protein always acted somehow as a "spacer" between the plate and the peptide making it more accessible for the antibody.
Hmmm....but it is worth trying as it seems much less effort than the conjugation thing...
Which binding/coating buffer do you use?
For proteins we usually take Carbonate buffer pH 9. Should be also sufficient for peptides?!
Thank you!