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

brett brett at BORCIM.WUSTL.EDU
Mon Dec 18 15:12:28 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 K. Troyer, PhD.
>University of Maryland School of Medicine
>Department of Biological Chemistry
>jtroyer at umabnet.ab.umd.edu
>*****************************************

An even simpler solution is to titrate the amount of Ab used. Most IP's are done
in a vast excess of Ab that will be ppt'd with the Protein-A. I take a
fixed amount of extract, and setup IPs with a dilution series of Ab (try 
the volume you are using down to 1:1000 of this amount). As you use less Ab,
the h.c. smear will disappear. At a much lower [Ab], the amount of signal will
decrease, of course. A dilution in between will be your working concentration
for future IPs. Needless to say, this also saves on valuable antibodie$.



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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