MAbs and Western Blot.

Ian A. York iayork at
Tue Jan 23 13:00:58 EST 2001

In article <D9gb6.2754$1K1.26031 at>,
Joe Chandler <jchandler at> wrote:
>The immunogens supplied to us are varied.  Most often, they are peptides or
>fusion proteins representative of a cell constituent.  I think from what I
>have gathered thus far, that the denaturation of the target molecules
>changes it sufficiently enough to make the monoclonal binding antibody not
>recognize the same site as it did when the molecule was in its native form.
>The answer for us is to generate more fusion products in the hopes that we
>will find one that will recognize a sequence that is unchanged during the
>denaturation process.

It's a little (but not terribly) surprising to me that mAb raised against 
peptides work against native, but not denatured, antigen; I'd tend to 
expect the reverse, because peptides will usually not be in a "native" 
conformation (that is, with respect to the full-length protein).  With 
fusion proteins, it's less surprising, because there is a good chance that 
the fusion will be more or less in a native conformation.

One approach might be to take your fusion protein and denature it before 
raising the antibodies:  boil it in the presence of  DTT and SDS, if you 
can clean it up well enough afterward to put it safely into the animal.  

If you're designing peptides, then the shorter the better (within limits, 
of course); it's conceivable that a long peptide might partially fold.  In 
principle, I think, a hydrophilic stretch would be a better choice than 
hydrophobic, because hydrophobic streches are more like to be be bound to 
the membrane and inaccessible.  (Of course, they're also more likely to 
be hidden inside the folded protein as well.)  

My experience with this is very limited, so this is mostly theory 
speaking, not observation.

    Ian York   (iayork at  <>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England

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