sandrine.vessillier at caramail.com wrote:
> Hi,
> I actually try to purify 2 proteins by gel filtration, one is 25KDa and
> the other one 15 KDa. SDS PAGE analysis indicates the first pic to be
> eluted is the 15KDa protein and I can not see the 25 KDa protein. Do you
> think it is retained on the column ?(I use 6M guanidine and 0.5M NaCl
> in tris buffer pH8 for loading and separation). Do you have some
> suggestions?
Superdex 200 is probably a bit large-pore for this application. You will
get better resolution with Superdex 75.
Guanidine is a pain in the neck during later stages like electrophoresis
or ion exchnge chromatography. Replace with urea, if your protein
allows. Also check, wheter or not you actually need the denaturant in
the first place.
At which wavelength do you detect the proteins? 280 nm only works if you
have enough aromatic residues present. Alternatively go for the peptide
bond.
