dbell at qnis.net
Fri Jan 18 12:33:32 EST 2002
On Thursday, January 17, 2002 7:37 PM, D.K.
[SMTP:dk at no.email.thankstospam.net] wrote:
: dbell at qnis.net (dbell) wrote:
: >On Thursday, January 17, 2002 7:35 AM, D.K.
[SMTP:dk at no.email.thankstospam.net]
: > wrote:
: >: 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
: > with
: >: >> >a smear down the lane instead of a tight, compact band. I know
: >: >> >protein isn't degraded and that the antibodies are specific to the
: > protein
: >: >> >and I have remade all my buffers. The only thing I can think of
: > might
: >: >> >be causing a problem is the acrylamide. I read that acrylamide can
: > up
: >: >> >to 9 months unopened and mine has been open for about 18 months,
: >: >> >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
: >: >> 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
: >: Because it is clearly not important for SDS-PAGE :-)
: >: DK
: >For those of us that are ignorant - please explain what you seem to
think is so
: > clear!!
: 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.
: 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.
But the separation is still due at least in part to size differentiaitons
and, since acrylamide / linker polymerization make up the gel, if you have
poorly polymerized gels, it will effect migration - even on SDS gels.
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