You received some excellent advice from Greg and Gautham on how to handle
your peptide. My extremely pragmatic advice is to pick another sequence.
Even if you use the advice to successfully make a good conjugate, the
disulfide-sulfhydryl question isn't going away. If there is any chance
that those Cys residues are bonded to Cys in other parts of the polypeptide
chain, you will likely get an antibody that won't be useful. If they are
in disulfides and you covalently modify the cysteines, the peptide will
handle well and probably elicit an antibody, but will it detect the native
protein? If the cysteines are present as free sulfhydryls, then
modification may also lead to an antibody that won't detect the protein
itself. If you leave them unmodified, chances are the peptide and its
conjugate will form multimers, and so on. If from any of these trials you
might obtain an antibody that sees the protein in an immunoblot, its
affinity and specificity may be low, and it will likely not work in useful
applications such as immunoprecipitation and confocal microscopy. This may
seem depressing, but I'm sure your real goal is to get a terrific antibody
to do some key experiments needed for an exciting paper in an excellent
journal.
Best regards,
Ruth
At 02:11 PM 6/11/98 -0500, Bao-Shiang Lee wrote:
>Hi everyone,
>
>I am raising an antipeptide antibody against a peptide (CGSCVDGRCCTP). The
>synthetic peptide is couplied to KLH using glutaraldehyde. The rabbit
>failed to produce any antibody. We think that the Cs may be the reason
>for this failure. Please give me some suggestions so that I can overcome
>this problem. Best Wishes.
>
>Bob Lee
>
>
>