We are having some major (and very strange) problems with our Western blots
and I was hoping someone has some suggestions to solve the problem.
When we run Western blot gels the proteins and marker go through the stacking
the resolving gel and start to resolve nicely. After an hour or so of running
the gel the
high molecular weight proteins and markers start to disappear from the top
By the time the dye front has reached the bottom of a long gel (16cm gel) the
marker has started to disappear or become very blurred.
If we transfer these gels there are no proteins on the top of the membrane and
looks like a transfer problem (although we know the problem occurred before
Since this problem began we have changed everything common to all people
acrylamide, SDS and Laemmli running buffer). We have measured the pH of all
solutions and they all seem fine and we have recleaned all plates. We have
this problem with all percentage gels and it seems to be getting worse over
time (as if a
component of a buffer or something is slowly going off).
We all use different power packs for running gels and either run them at 30mA
or 50V overnight (both of these methods still lead to the same problem).
Any advice or previous experience with this kind of problem would be most