Lane broadening on protein gels

Craig Marshall craigm at sanger.otago.ac.nz
Wed Nov 24 17:46:57 EST 1993


Michael Holland (M.Holland at dwe.csiro.au) wrote:
> Hi gurus,
> We have problems with unacceptable lane broadening with our samples during
> SDS-PAGE. We have to solubilize our material by heating at pH 10 and 70 C
> for 10 minutes. The pH is adjusted to 7 with acetic acid and the samples
> Speedy Vaced to dryness. The residue is taken up in 20 ul of standard Laemmli
> sample buffer at pH 6.8 and loaded onto Hoeffer mini gels. From the colour
> of sample buffer, which contains bromophenol blue, the pH remains unchanged.
> When the gels runs everything looks great but on staining the bands are 2-3
> times the width of the lane. The MW standards run fine as do other gels with
> different samples. Clearly the problem seems to be in the sample
> preparation. Any hints?????
> Michael K Holland PhD
> CSIRO, Wildlife & Ecology
> PO Box 84, Lyneham ACT 2602, Australia
> Fax: 61-6-242-9242  Phone:61-6-242-1793
> mholland at dwe.csiro.au

There are two things that come to mind: You don't say what you used to
adjust the pH to 10. Although the acetic acid is volatile, the salt
produced may not be and so excess salt may be causing the band
broadening. One thing that I have found to be useful is the
substitution of half the tris in the stacking buffer by imidazole.
The pK of tris at about 8.3 means that it does not buffer at 6.8,
whereas imidazole does, so this substitution controls the pH of the
sample and stacker better and makes it less susceptible to the effect
of excess salt and altered pH, both of which can give the symptoms you
describe. A reference for this is Ritterhouse, H. and Marcus, F.
(1984) Peptide mapping by polyacrylamide gel electrophoresis after
cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl
sulfate-containing buffers Anal. Biochem. 138:442-448
 

--
        Craig Marshall          	craigm at sanger.otago.ac.nz or
        Biochemistry Department 	bioc07 at otago.ac.nz
        University of Otago     	Phone 64 3 479 7849   	
        P.O. Box 56             	Fax   64 3 479 7866   	
        Dunedin, New Zealand                                   	



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