Western Blot Problem

Martin Blankfard Mblank at kpl.com
Wed Jun 21 13:03:55 EST 1995


In article <3s83nm$kuu at news.ccit.arizona.edu>,
   valeryt at ag.arizona.edu (Val Thompson) wrote:
>A student in my lab has been running a lot of western blots in order 
>to map the epitopes of several of our monoclonal antibodies.  She is
>running samples of antigen (a protease) that have undergone limited 
>digestion by  trypsin and chymotrypsin in addition to the native protein
>and its auto-proteolytic fragments.  We have discovered a very strange
>phenomenon (at least to us).  The antibodies no longer recognize samples
>that have been stored frozen at -20C in SDS sample buffer for several
>weeks.  We normally aliquot the samples so that they will only be thawed
>and refrozen 4 or 5 times before the sample is used up.  When fresh 
>sample is prepared the antibodies bind to more of the fragments than
>if "old" sample is used.  We discovered this when the student did a 
>WB loading freshly prepared trypsin and chymotrypsin digested samples, but 
>old autolyzed and native protein.  She stained the membrane after transfer 
>and the protein bands in all samples were clearly visible, but the Ab did
>not bind to the autolyzed or native proteins, but did bind to 
>fragments in the tryptic and chymotryptic digest.  She repeated the same
>blot using freshly prepared autolyzed and native protein and then the Ab
>did bind as expected.  We have since discovered that this happens with
>3 other of our monoclonal Abs.
>
>So the question is "What's going on here?"  Can the epitopes become masked
>during storage?  Do they somehow deteriorate so that the Ab can't bind
>(we don't see any "extra" bands in these samples)?  Has anyone else had
>a similar experience?  I've talked to several people in my department and
>no one has a really good explanation.  I'd appreciate hearing from 
>anyone who may have some insight into this matter. 
>
>Thanks,
>
>Val Thompson
>Muscle Biology Group
>University of Arizona
>Tucson, AZ  85721
>valeryt at ag.arizona.edu 


SDS often destroys antigen secondary and tertiary structure.  Monoclones are 
frequently directed against secondary and tertiary structure NOT primary 
structure. For western blotting polyclone (specifically affinity purified 
polyclones) are your best bet for probing western blots.


Martin Blankfard
Kirkegaard and Perry Laboratories
2 Cessna Court
Gaithersburg Maryland
Mblank at kpl.com



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