polyclonal antibodies: help!

John K. Troyer jtroyer at umabnet.ab.umd.edu
Mon Sep 25 10:32:35 EST 1995


On 25 Sep 1995, Giorgio Spagnol wrote:

> Dear fellow researchers,
> I need to raise antibodies to a type 1 membrane glycoprotein of 100 kD.
> Since I want these Ab's to act against the extracytoplasmatic peptidic
> moyety, and I do not have sufficient quantity of purified antigen, I
> tought about using a sinthetic peptide as an immunogen.
> Do you have any idea how to choose it, given that the whole sequence of
> the protein is known?
> Thanks in Advance.
> Giorgio.


Giorgio:

There is a good computer program out, available in packages such 
as PC Gene, which was written by Hopp, T.P. and Woods, K.R. (Mol. Imm. 
20: 483-489, 1983).  This program picks antigenic peptides based on 
relative hydrophylicity, under the assumption that hydrophobic peptides 
will be buried in the interior of the tertiary structure, while the 
hydrophylic peptide stretches will be exposed on the outside. 

My experience with the generation of peptide antibodies to native 
proteins has been mixed.  Several suggestions I would make are:

1) Make several different peptide antibodies since the programs give you 
just a best guess about which peptides will be exposed and do not take 
into account any post-translational modifications.  

2) MAPS (Multiple Antigen Peptide System) peptides give good results and 
negate the need to covalently crosslink the peptides to carrier 
molecules.  The only negative I have found is that some antibodies are 
produced against the lysine core, although the manufacturers claim this 
does not occur.  Most facilities which make synthetic peptides can now 
make MAPS peptides and can give you further information.

3) When chosing peptides, take into account possible glycosylation 
sites.  If there any potential sites within or next to the peptide of 
interest, do not use the peptide to make antibodies. 


Good luck

John

**************************************************
John K. Troyer, PhD
University of Maryland School of Medicine
Department of Biological Chemistry
(410) 706-7518
jtroyer at umabnet.ab.umd.edu
**************************************************




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