Weird Western Blot (circular streaks)

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Jul 20 10:07:33 EST 2000


Hi Soenke,
We always assemble the blots in a "semidry" way, not "underwater" as you
said. I feel it is much easier to put the gel and the membrane in the
desired place in that way. I´m not sure what kind of artifacts Bernard
refers to (?). I just do it for practical reasons.

As to the original post: May the membrane has creases (that take up the
ponceau red, but do not really disturb your immunoblotting result). Or
the blot sandwich is not completely flat (perhaps the sponges or the
blotting cassette are dirty or distorted). Or (if you use a semidry
blooting apparatus), the electrodes are slightly bent and do not make a
good contact to the sandwich.


Hope this helps, 
Gruesse aus Konstanz (aber bald auch in Hamburg)
Frank



soenke behrends wrote:
> 
> Dear netters,
> 
> does Bernhards comment here indicate that it is not good
> to pack the sandwich with the gel and the nitrocellulose
> 'underwater' in the transfer buffer? I have been trained
> to do so and have never questioned it or tried to pack it
> 'outside' and soak the whole sandwich afterwards. (I too
> am speaking of the Biorad Minigel system).
> 
> >I hope you don't soak your gel before transfer.  It's a
> >waste of time and can lead to artefacts.
> 
> Thanks for your time and help
> Soenke






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