Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Sun Feb 29 06:46:57 EST 2004

miaoqf at sina.com wrote:

> I expressed a protein in E.coli sucessfully  and the DNA sequence is
> right. The theoretical MW is 25KD. But after its purification, the MW is
> 29-30KD in a reducing-SDS PAGE. More surprised me is the MW is 25KD when
> I run it in a non-reducing SDS pAGE. I don't know the reason. Who can
> help me ?

MW determination by SDS-PAGE is based on the idea that proteins on
average bind 1.4 g of SDS per g of protein, resulting in a constant
charge/mass ratio and hence constant acceleration in an electric field.
Also assumed is that proteins under denaturing conditions all assume a
more or less similar shape. Hence the interaction with the gel matrix,
and thus Rf values, depend only on size.

Under non-reducing conditions the protein may assume a different
conformation, due to the presence of disulphide bonds.

Very hydrophobic proteins can bind more SDS than average, resulting in
higher charge/mass ratio. A typical example is Na/K-ATPase
alpha-subunit, which has an apparent MW in SDS-PAGE of 86 rather than
112 kDa.

Glycosylated proteins contain additional negative charges from
carbohydrates, which can influence migration. The number of charged
sugar residues is highly variable, resulting in smeared bands.

Proteins with a large number of charged amino acids will run to
positions of higher or lower MW, depending on polarity. A typical
example are histons.

The latter two problems may be circumvented by using a positively
charged detergent like CTAB. I have published such a system in Anal.
Biochem. just a year ago. But a precise determination of molecular
weight would require other methods than electrophoresis (e.g. analytical
ultracentrifugation, mass spectrometry).

More information about the Proteins mailing list

Send comments to us at biosci-help [At] net.bio.net