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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Western blot problem -disappearing proteins!
 

jscorah wrote:


Have you tried to use coloured marker proteins (rainbow-markers or the
like) to watch what happens during the run?

Where in relation to the front is the 75 kDa protein, i.e. which
percentage gel are you using? Have you considered using gradient gels?
Resolution at the bottom of the gel is quite low unless you use
gradients.

Does the problem also occur with gels bought ready-to-use?

Do you store the gel o/n after casting it to make sure that all radicals
formed during polymerisation are inactivated?

Is the sample free of salts? Has the acrylamid been stored over ion
exchanger to capture any acrylic acid formed during storage? Measure the
conductivity of sample and gel mixture (before polymerisation) and of
the running buffer. Increased conductivity will lead to band smearing
and gel heating (even boiling!). There are nice pen-shaped conductivity
meters available which are not very expensive and accurate enough for
this purpose, the type I use requires only about 20 ul of sample.

Western blot problem -disappearing proteins!
 

"jscorah" <[Only registered and activated users can see links. Click Here To Register...]> wrote in message news:41BA3E9D@neo...


We had a similar problem with SDS-PAGE some years ago. The low-molecular
weight proteins were not present or very blurred in the last third part of
the gel. We found out that the SDS were simply not pure enough. Your problem
could be related, so you might check the purity of the SDS. The specified
amount of C12 alkyl should be no less than 98%.
Good luck
Jens

Re: Western blot problem -disappearing proteins!
 

hi ! Iam facing the same problem my protein is arnd 10kDa and no bands appear below 14kda band after staining with ponceau. Initially even I thought it to be a transfet problem but when I coomassie stained the gel bands were again not present below 14kDa band ,I even tried pre stained marker have u tried it n hw do u observe the disappearing of bands while the gel is running.

Re: Western blot problem -disappearing proteins!
 

Do you use the same box for transfer as for running your gel? A little bit of residual methanol might gradually strip your proteins of SDS.

Re: Western blot problem -disappearing proteins!
 

How early do you make your gels?