Migration of protein in SDS PAGE gel

Giovanni Maga maga at vetbio.unizh.ch
Wed Dec 20 08:46:53 EST 1995

In article <4at61j$gfj at nuscc.nus.sg>, mcbthy at leonis.nus.sg (Tang Hsin Yao)

> Hi! I have a problem here and I hope somebody out there can help me. I
> have a protein with a predicted molecular mass of 150 kD. However when I
> ran a SDS PAGE gel, it migrates at the size of 200 kD. Is there any good
> explaination for this? I have read somewhere that certain proteins rich
> in certain amino acid residues will move slower than their predicted
> size. Is this true? I hope someone will point me to the references regarding
> this matter.
>         Thanks a lot in advance.

It's known that strongly acidic proteins (i.e. with pI below 5) usually
run slower in SDS-PAGE, due to the fact that SDS and the protein are both
negatively charged at the buffer pH, thus the electrostatic repulsion
interfere with protein denaturation by SDS. Another possible explanation
is postranslational modification (i.e. phosphorylation), if you are
purifing your protein from tissue or from an expression system that allows
also postranslational modifications (i.e. eukaryotic proteins in
baculovirus for example or yeast proteins in yeast). To have a quick check
of the net charge of your protein, you can load it on a weak anion
exchanger equilibrate at pH 6.0 and wash it with 150 mM salt. If it binds
and does not elute, it means that at pH 6 it is still enough negatively
charged to interact with the matrix specifically (I would bet then that
its pI will be at least 1 point below), so you can be quite sure at the
basic pH you are using for the gel it is heavily negative.
Hope it helps. Giovanni.

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