gel filtration of glycoproteins

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Thu Feb 6 19:32:45 EST 2003


> Following problem:
> I've got a protein of about 40 kD (according to its amino acid-sequence)
which is glycosylated, when I pass it on a gel filtration column it gives me
a very broad and flat peak (well, probably one wouldn't call it a "peak"
anymore) somewhere in the range between 70 and 100 kD.
>
> Question:
> If you look at a glycosylated protein in general, would you expect such a
behaviour or should it still run as a discret peak? How much does the
glycosylation affect the apparent MW of a protein when doing size exclusion,
is it likely to increase the size by a factor of 2 or 3, or is this more a
hint for dimerisation?

0) what does MS have to say - what is the real m.w. of the protein ? if you
can't do MS at least run a denaturing gel - how many bands do you get, and
how smeared ? Can these bands be changed via treatment with deglycosylating
enzymes e.g. PNGase F ?

1) did you express the protein in its native host or in a foreign host ? if
it's a heterologously expressed protein, you will frequently have unusual
and irregular glycosylation patterns, often leading to abnormally high sugar
content and heterogeneity of the sample due to irregular runaway
glycosylation.

2) generally speaking, extensive glycosylation does indeed change the
behavior of the protein on size exclusion. Usually, as long as you have
homogenous species, your peaks would be reasonably sharp, however if you
have heterogeneity than all bets are off - anything can happen.


Good luck,

A.G.E.





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