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
