Background staining due to epitopes to Keyhole limpet hemocyanin

Thomas R. Anderson babco at ix.netcom.com
Sat Aug 15 00:48:42 EST 1998


In <6qupo1$meb$1 at nnrp1.dejanews.com> pleuronectes at my-dejanews.com writes: 
>
>I have been using a keyhole limpet hemocyanin conjugate to make
antibodies to
>winterflounder type I serum anitfreeze protein.  We have been raising
the
>antibodies in Rabbits and using the antisera for both
immunohistochemistry
>and protein dotplots. The antisera has also been used for western
plots. 
>Frequently we see contradicting results between western blotting and
>immunohisto.  I am wondering if some of the results could be due to
>background staining of epitopes to KLH. Insitu hybridization will
sometimes
>backup the result and sometimes not?
>
>Anybody out there have similar problems?  Any comments will be
appreciated, I
>am trying to finish up my thesis experiments.
>

Why don't you try cleaning up your antibody prep?  Two ideas:  first,
adsorptively remove the antibodies against KLH by passing the crude
antibody over an affinity resin containing KLH.  That should remove
most of the antibodies against KLH, and if indeed the problem is caused
by antibodies in KLH, the problem would be ameliorated (or at least
diminished) by this strategy.

A second approach would be to affinity purify the antibodies against
the peptide by making an affinity resin bearing the peptide you used in
the immunogen but without the KLH.  Pass the crude antiserum over the
column, wash away the non-adherent antibodies with buffer, then elute
the specific antibodies with a drop in pH.

Better still, do both procedures.

If you can't find protocols to do this, feel free to email me, and I
will point you in the right direction.

Good luck with your studies.

Tom Anderson



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