moropeza at MAIL.MTY.ITESM.MX wrote:
>> My name is Yuriana Oropeza, and I am a master degree student in
> Mexico. I am trying to produce monoclonal antibodies using a peptide
> as antigen. I was coupling the peptide to BSA and my questions are:
> I am not sure if the coupling was complete, do you know any protocol
> easy to perform to ensure this?
> I want to screen with ELISA, and I want to know the possibility to
> coat the peptide to an ELISA plate. Peptide molecular weight: 1430
> Do you know any protocol to do this?
> Thanks for your help.
The technique used to determine the coupling efficiency of peptide to BSA
depends on how the peptide was conjugated. The common method of coupling
through terminal cysteines makes coupling efficiancy determination simple. Just
do an Ellman's assay (dtnb).
Coating a peptide on an ELISA plate has a few inherent problems. To begin with,
the interaction between plain polystyrene plates and small peptides can often
cause degradation of the peptide. You may want to try a different aproach such
as biotinylating the peptide and immobilizing it on a Neutravidin coated ELISA
plate. This has the added benefit of inserting a speacer arm between the plate
and the peptide, htereby increasing antibody recognition.
Tom Brotcke/Pierce Chem.
