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[Protein-analysis] Re: Protein MW deviation

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Tue Sep 26 04:09:45 EST 2006


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}
}



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