Cracking 16% acrylamide SDS-PAGE gels
(by ipek4477 from gmail.com)
Sat Jul 4 13:53:18 EST 2009
I would be grateful if you could share these formulas against cracking gels.
I usually work with 12.5% polyacrylamide gels, I hardly have a problem; but
it would be nice to try some other recipe.
Thanks in advance,
On Sat, Jul 4, 2009 at 8:03 PM, <methods-request from oat.bio.indiana.edu> wrote:
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> 1. Re: Cracking 16% acrylamide SDS-PAGE gels (Nick Theodorakis)
> Message: 1
> Date: Fri, 3 Jul 2009 14:19:57 -0700 (PDT)
> From: Nick Theodorakis <nick.theodorakis from gmail.com>
> Subject: Re: Cracking 16% acrylamide SDS-PAGE gels
> To: methods from net.bio.net
> <542565ee-ce63-41c8-85fc-5116f39e19a9 from n11g2000yqb.googlegroups.com>
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> On Jul 2, 3:05 pm, "Paul J. Phelan" <Paul.Phe... from tufts.edu> wrote:
> > I think this is a trivial problem, but I have been trying to detect and
> > resolve a cyclic peptide of mol. wt. 1,300 from a bacterial extract. I
> > am using a peptide gel recipe for SDS-PAGE based on the Schagger + von
> > Jagow Tris/Tricine system, that uses a 16% acrylamide resolving gel, a
> > 9.5% acrylamide spacer gel, and a 5% acrylamide stacking gel. I'm
> > having trouble detecting the peptide (it contains a 35S-Met), but my
> > question is related to a problem we are having with the gels cracking
> > after they are dried on a gel drier and then frozen at -80 C before
> > being developed on x-ray film. Most of the time, the gels are cracked
> > after drying, and freeze/thawing just makes the cracking worse.
> > I think the gels are cracking just because they are more brittle at a
> > high acrylamide concentration, but does anyone have any fool-proof
> > tricks for drying thin (0.75 mm) 16% acrylamide mini-gels without
> > cracking them?
> > Any words of wisdom much appreciated,
> > Paul Phelan
> Unless you're doing fluorographic detection, you don't need to freeze
> the gel during exposure.
> I use a little=known gel recipe from Blattler et al (1972) that uses a
> variable acrylamide:bis ratio that seems to give gels with much better
> physical properties, such as resistance to cracking. The reference is:
> Blattler DP, Garner F, Van Slyke K, Bradley A. Quantitative
> electrophoresis in polyacrylamide gels of 2-40%. J Chromatogr.
> I also have a spreadsheet with formulas I can send you if you want to
> try it.
> Nick Theodorakis
> nick_theodorakis from hotmail.com
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