Protein electrophoresis: pH of sample buffer

Helge Weissig helgew at LJCRF.EDU
Tue Nov 9 22:19:43 EST 1993


>Hi
>This is the sort of embarrasing question that when you are asked by a
>graduate student you feel you should know the answer but don't:
>Why is the ph of the sample buffer in Laemmli systems 6.8 when the stacking
>gel it is entering is 8.9?? Can it be altered to be alkaline?
>Michael K Holland PhD
>CSIRO, Wildlife & Ecology
>PO Box 84, Lyneham ACT 2602, Australia
>Fax: 61-6-242-9242  Phone:61-6-242-1793
>M.Holland at dwe.csiro.au

it sure can't. The reason being, that the lower pH of 6.8 is about the same
as the pI of Glycin (Laemmli gels are run with Tris-Glycin-SDS buffer). So
Glycin is rather uncharged at that pH and starts trailing behind the
samples, while the chloride ions run ahead. This creates a zone of lower
conductivity (=steeper voltage gradient) which causes the protein samples
to stack (that's why the upper gel of this "discontinous" system is also
called stacking gel). The higher pH in the lower gel (= resolving gel)
causes Gly to loose a bunch of H+ (statistically spoken) to the OH- rich
environment and Gly joins Cl to run ahead thereby causing the voltage
gradient to disappear. Now the proteins are separated due to the sieving
effect of the polyacrylamid matrix.

And don't you think i knew that all from my not so long ago undergrad exam
(which is rather something complex where i come from). i mixed it all up
and had to look it up in the Maniatis (pg 18.47) myself.

cheers,
helge

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