Cracking 16% acrylamide SDS-PAGE gels

[Nick Theodorakis]

On Jul 2, 3:05=A0pm, "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. =A0I
> 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. =A0I'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. =A0Most 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=3Dknown 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=9640%. J Chromatogr.
1972;64:147=96155.

I also have a spreadsheet with formulas I can send you if you want to
try it.

Nick

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