Making antipeptide antisera is a crap shoot. Each peptide will cost you $500
to make and $500 to shoot in to bunnies. Its worth it if it works though. For
my cDNA (unknown protein) I did the following:
1) do sequence analysis (there are lots of WWW sites for this; dont wast your
time with looking for one specific program) and look for regions prediceted to
be (A) solvent accessible (B) antigenic (C) not predicted to fom alpha, beta or
any other structure.
2) pick a peptide (a 20mer is good b/c you get several potenital epitopes
within it) containing one proline (to decrease potential conformiataional
changes between the peptide and the native structure of the protein
3)coulple it to MAPs or KLH and put it into rabbits
Test the antisera by several ways (ELISA using the peptide, IP, Western...)
I designed 3 20mers in a 300 AA protein. All three sera are excellent.
In article <hodges-1312951649390001 at cormus.ibp.u-psud.fr>, hodges at psisun.u-psud.fr (Susana Galvez) writes:
> Hi everybody:
>> We are interested in purifying a really very very unstable plant
> protein; fortunately, we have found the corresponding cDNA and the protein
> has been produced (we hope so!) in E. coli. Our problem is that we can't
> measure the enzymatic activity of this protein in the bacterial extract
> (we guess it's too unstable), and we can not detect by SDS-PAGE a protein
> So, we are thinking to use another strategy: to raise antibodies
> against several peptides (deduced from the cDNA sequence).
> But, which ones??
> My question is: Can anyone send me a program capable of suggesting the
> "more probable" antigenic sites (antigenic peptides) of a protein from the
> deduced amino acid sequence?