I am seeking advice for a researcher who is doing electroblot onto PVDF of
his protein--using Gelman Biotrace PVDF. He has had success with getting
great transfer of proteins and we generally get good sequence data--until
recently. We are wondering what is degrading in his electroblotting
system. He suspects that now proteins are being N-terminally blocked
during the gel run and or transfer. I've suggested that he make up fresh
solutions, but thought it would be helpful to get comments from those of
you who run gels.
First of all, what would be a good protein to run as a standard that would
be sensitive to N-terminal blockage--to use as a control during the run
and transfer?
Have you found old solutions of transfer buffer, acrylamide (I believe
that answer to this is a yes) to block proteins?
He is using a stock of thioglycolic acid that is now two months old--could
that be a factor?
These proteins are not seeing urea and we have gotten sequence data from
them before.
We are aware that you can get "new" protein bands from old stocks of SDS
and 2-BME--do these stocks ever block proteins?
Also, he does boil the samples before application to the gel--my memory is
that this can cause N-terminal blockage on some proteins (which N-terminal
amino acids are most susceptible to this?)
Thanks for your advice/help,
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR