In article <80f0t5$5ae$1 at nnrp1.deja.com>, Kresten <kresten at my-deja.com> wrote:
:In article <pxpst2+-0611991043130001 at pelli.pathology.pitt.edu>,
:pxpst2+ at pitt.edu (Peter) wrote:
:> In article <3821AEEE.9EFC3D58 at sheffield.ac.uk>, Lee Hunt
:> <L.hunt at sheffield.ac.uk> wrote:
:[snip]
::> Always remember that PAGE does not seperate purely by mass/size.
:> Seperation is by the charge to mass (m/z). Alter this and and you
:> will have abherent migration.
::This is why we include SDS in our PAGEs. If it were so that proteins
:bound a number of SDS molecules proportinal to their mass and that this
:number was so large that the proteins own charge would be negligable
:compared to the charges that SDS bring, then all proteins in SDS would
:have approximately the same m/z. The idea is then, that you separate on
:m (or rather volume or some other structure parameter) using the sieving
:effect of the PA gel.
All is right but it's worth remembering that nothing is ideal under the moon.
In case of SDS PAGE, the number of SDS molecules bound per MW is
_not_ the same for all sequences. Thus, the mobility. The bottom line is
that SDS PAGE as a criterion for estimation of MW is better than
gel filtration but can still be very far from true MW based on sequence -
even when corrected by known posttranslational modifications. Some
examples: phosphorylation (32+3x18 Da) at single site can upshift the
band by as much as ca 5 kDa, calmodulin runs 18 or 22 KDa depending
on the presence of Ca++, myristoylation of ARF1 upshifts the band on
a gel, but myristoylation of ARF6 (95% homology, but a very different pI)
results in downshift.
Dima
