dk at no.email.thankstospam.net
Fri Jan 18 23:38:36 EST 2002
dbell at qnis.net (dbell) wrote:
>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.
Your typical "bad" acrylamide with high acrylate content polymerizes
quite well. Since in this case the diagnosis appears to be gel not
polymerizing properly and different acrylamide working well, I'd think
that "acryalamide" probably means "mono and bis _mixture_".
Then I tend to think that the "bad" arcylamide/bis-acryalamide
mixture simply was not done in right proportions.
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