Some proteins are kinetically stable and resistant to SDS when the sample is not heated. I'm looking for a more denaturing detergent than SDS.
Hi there,
Usual cracking buffer supplemented with 8M Urea is definitely more potent toward difficult cases and fully compatible with SDS/PAGE (whereas GdmCl is a pain because it precipitates in presence of SDS). The only constraint is that you have to load every single well of your gel with it (including MWM) in order to avoid lateral diffusion of urea during running. I use the following composition: 40 mM Tris–HCl, pH 6.8, 8 M Urea, 5% SDS, 0.1 mM EDTA, 1%b-mercaptoethanol, 0.04%Bromophenol Blue.
CTAB is not really more denaturing than SDS, but can bring some proteins into solution that resist heating in SDS, sometimes while maintaining their enzymatic activity for detection in zymograms. It can be used for all electrophoretic methods (1D, 2D, blotting, electroelution, ...) and staining methods (CBB, RuBS, Ag, stains-all, Feulgen...), see doi:10.1016/S0003-2697(02)00639-5, 10.1007/978-1-59745-542-8\_14.
For electrophoresis in the presence of urea I'd go one step further than Dominique Liger and include it in the gel too. Like Dominique, I'd advice against GdmCl: electrophoresis works the better the lower the ion concentration is.
I agree with Dominique, with the only caveat that you should be careful not to heat urea for too long, as it will form cyanate, which is ionized and will mess up the stacking and migration of the samples. However, unless you are working with hyperthermophiles, most proteins will be denatured by boiling for 5 min in regular Lämmli sample buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% mercaptoethanol, 10% glycerol and 0.04 % bromophenol blue
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