dk at no.email.thankstospam.net
Thu Jan 17 22:36:44 EST 2002
dbell at qnis.net (dbell) wrote:
>On Thursday, January 17, 2002 7:35 AM, D.K. [SMTP:dk at no.email.thankstospam.net]
>: Michael Witty <mw132 at mole.bio.cam.ac.uk> wrote:
>: >> sweiss at icr.ac.uk wrote:
>: >> >
>: >> >I am having major problems carrying out western blots. I ALWAYS end up
>: >> >a smear down the lane instead of a tight, compact band. I know that my
>: >> >protein isn't degraded and that the antibodies are specific to the
>: >> >and I have remade all my buffers. The only thing I can think of that
>: >> >be causing a problem is the acrylamide. I read that acrylamide can last
>: >> >to 9 months unopened and mine has been open for about 18 months, do you
>: >> >think this could be affeecting my gels?
>: >> >
>: >> Could be many things but don't blame it on acrylamide. Here we use
>: >> some terrible junk given in large quantity for free about 8 years ago and
>: >> it works fine for SDS-PAGE/westerns. Acrylamaide quality is only
>: >> important for non-SDS applications.
>: >.. . . why is acrylamide quality only important for non-SDS applications?
>: Because it is clearly not important for SDS-PAGE :-)
>For those of us that are ignorant - please explain what you seem to think is so
Like I said, for many years I was using ugly yellow crap and have done
many beautifully looking gels, westerns and even obtained crystal clean
mass spec sequencing results using it. Pics available on request :-)
Why? Just a guess: "Bad" acrylamide has a high acrylic acid content.
Thus, gels made with it will have plenty of residual negative charges. This
is clearly very bad for non-SDS applications (electroosmosis, ionic
sorbtion, etc). None of this really matters in SDS-PAGE protocol because
it is done in high capacity buffer, separates essentially based on the
size charge of SDS-protein complex, with complexes bearing large
negative charge and thus moving along very fast.
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