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Western blot problem -disappearing proteins!

Western blot problem -disappearing proteins! - Protocols and Methods Forum

Western blot problem -disappearing proteins! - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 12-13-2004, 02:21 PM
Jayakumar, R
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Default Western blot problem -disappearing proteins!



Try changing the SDS apparatus. Looks to me like there is a linear
electric filed perpendicular to your gel developing.


Jai
-----Original Message-----
From: [Only registered users see links. ] [mailtowner-methods@hgmp.mrc.ac.uk]
On Behalf Of jscorah
Sent: Friday, December 10, 2004 5:04 PM
To: [Only registered users see links. ]
Subject: Western blot problem -disappearing proteins!

Hi,
We are having some major (and very strange) problems with our Western
blots
recently
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
gel into
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
downwards.
By the time the dye front has reached the bottom of a long gel (16cm
gel) the
75kDa
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
it almost
looks like a transfer problem (although we know the problem occurred
before
then).
Since this problem began we have changed everything common to all people

(including
acrylamide, SDS and Laemmli running buffer). We have measured the pH of
all
our
solutions and they all seem fine and we have recleaned all plates. We
have
experienced
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
for 3hours
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
welcome!
Thanks.
Jenny

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