overexpressed protein sometime gives two bands

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Tue Jan 15 21:52:17 EST 2002


Dear Tina,

You can't know for sure until you determine accurate molecular weight of the
two sub-species. The easiest and the most reliable way to do so would be to
submit them for MS. If your difference is really that large (5 kDa is
enormous!) then you will definitely see it clearly and probably will be able
to determine what's going on. Reasons for a 5 kDa difference can be as
simple as proteolysis and as complicated as shock-induced co-expression of
various bacterial proteins, rare codons, and protein modifications which can
give rise to an 'apparent' m.w. difference without actually casuing a 5 kDa
real weight change. A classical example of the latter would be
phosphorylation since SDS-PAGE of a mixture of phosphorylated and
nonphosphorylated protein will often (almost always, in fact) run as two
bands due to charge difference.

A.G.E.

"user" <q6252397 at fernuni-hagen.de> wrote in message
news:a225sc$nf2$1 at f1node01.rhrz.uni-bonn.de...
> Dear all,
>
> I'm overexpressing different proteins (phage, E. cloacae and C. freundii,
> with and without his-Tag) in E. coli.
> Usually I have a good expression and a single band during purification.
> I wonder why I sometimes get two band, which are there all the time during
> purification. They differ only very little in Mw. Probably much less than
5
> kD.
> I suppose, it is the same protein because it seems to bind on the column
> exactly like the "one-band-protein".
>
> Any explanations for this?
>
> TIA,
> Tina
>
>





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