DK wrote:
> In article <mailman.1077.1158617101.20007.proteins at net.bio.net>, "Sutk
> Nakasa" <kon_diew at hotmail.com> wrote:
> >Dear All,
> >
> >why sometimes the target protein size is bigger than the expected size when
> >running in SDS-PAGE? Some suggested that I should run my target protein
> >compared with un-prestained protein markers, or to make sure that there is
> >no cross-reaction once the immuboblot was performed. All of the above have
> >been tested but the results with bigger size of target protein than that of
> >expected one still a problem. Can anyone give me a clearer explaination or a
> >better way to verify or to confirm that I have the right target protein?
>> You have to realize that estimation of MW from SDS-PAGE is only an
> approximation that is valie for an average protein. There are pelnty of
> exceptions. If your protein happen to bind less SDS molecules or adopt
> conformation significantly different from average, you will always have
> "wrong" number. The classic example is phosphorylation where
> for some proteins single site modofication makes protein run 2-3
> "kDa" higher.
One might add that using several pore sizes (Ferguson-plot) gives more
precise results than using only a single gel type. This can be done
economically in a gel with transversal acrylamide gradient:
@article{Nis-89,
AUTHOR= {H. Nishizawa and N. Kita and S. Okimura and E. Takao
and Y. Abe},
TITLE= {Determination of Molecular Weights of Native Proteins by
Polyacrylamide Gradient Gel Electrophoresis},
JOURNAL= {Electrophoresis},
VOLUME= {9},
YEAR= {1989},
PAGES= {803-806},
LANGUAGE= {engl}
}
@article{Ret-88,
AUTHOR= {C. Retamal and J. Babul},
TITLE= {Determination of the molecular weight of proteins by
electrophoresis in slab gels with a transverse pore gradient of
crosslinked polyacrylamide in the absence of denaturing agents},
JOURNAL= {Anal. Biochem.},
VOLUME= {175},
YEAR= {1988},
PAGES= {544-547},
LANGUAGE= {engl}
}
