Re: Antipeptide antibodies
gsarath@unlnotes.unl.edu
Wed, 28 Jul 1999 10:00:33 -0500
Hi Latif: The paper using the his-tagged peptides was published in
Biotechniques in 1997 or early 1998. We have routinely made a number of
antipeptide antibodies, and have had limited troubles in purifying the specific
IgGs on an immobilized peptide column. This method using the His-Tag looks very
promising, but if I remember the paper correctly, the blots looked somewhat
patchy. I synthesized about 6 different 6X Histidine peptides for a user, but
have not heard back regarding their efficacy. In my limited experience, it
appears that the choice of the peptide antigen involves a large amount of luck,
and purification is almost a must. To illustrate this, I recently synthesized a
phosphopeptide antigen to an enzyme's active site. This was 22 mer peptide with
a short region of hydrophilicity. The peptide was coupled through a N-terminal
cysteine to KLH using MBS. The first bleed picked up only the phosphoenzyme in
blots. All subsequent bleeds yielded no observable cross reaction to anything.
Got me worried. We took the crude serum and ran it over an immobilized
phosphopeptide column, the bound fraction was eluted with Pierce Gentle Elution
buffer and bingo, we had really good antibodies. For three other antipeptide
antibodies, designed based on hydrophilicity etc, we essentially got nothing.
So the long winded answer is YES, but tread carefully!