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How to elute antigens without eluting Ab off Prot. A?

John K. Troyer, PhD. jtroyer at umabnet.ab.umd.edu
Mon Dec 18 10:14:59 EST 1995


On 18 Dec 1995, Noam Harel wrote:

> Hi,
> 
> 	I am performing immunoprecipitations on unlabeled nuclear
> extracts using non-covalently linked Protein A-antibody combinations.
> The problem is, when I run the precipitates on SDS-PAGE, then do a 
> Western blot, the secondary antibody on the W. blot causes the heavy
>  chains of the Abs used in the ip to create a 
> huge smear that covers up many of the potential bands I'd like to see.
> Is there any way besides covalently linking the Ab to the beads, 
> or besides eluting with competitor peptides, to keep the Ab from
> coming off the Prot. A beads when boiling in SDS-PAGE buffer?
> I heard something about elution using glycine, but can't find a protocol
> Thanks for anyhelp,
> 

Noam:

What is your aversion to covalently linking your Ab to the beads?  This 
is rather easily done and would solve your problem with very little effort.

I would be happy to send you a protocol if you require one.  Good luck!



John


*****************************************
John K. Troyer, PhD.
University of Maryland School of Medicine
Department of Biological Chemistry
jtroyer at umabnet.ab.umd.edu
*****************************************



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