his-tagged protein contaminate

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Fri Oct 5 21:10:29 EST 2001


> Hello!
> I purified 27kDa His-tagged protein (expressed in E. coli) on Ni-NTA
> column, 80kDa contaminate protein always presents in eluates. Could
> anybody recommend me some procedures to avoid the presence of this 80kDa
> protein? Thanks!!!
> Valeria.

Cheers,

How much of the contaminant is there ? 80 kDa versus 27 kDa - sounds perfect
for sizing, although nothing ever works perfectly :)

How do you perform your chromatography ? Do you load the material in 20-30
mM imidazole, with 300 mM salt ? What is the pH ? Can you sacrifice a bit of
your yield and use a slightly undersized column, so that it will become
saturated with the his-tagged protein ? Try running your columns in 20%
glycerol, play with salt and ph, try different modes of elution etc.

Also, beware, you most likely will also have a 21 kDa protein as a
contaminant, it is a well known proline isomerase which binds to Ni-NTA like
crazy. Again, using slightly undersized columnts sometimes helps.


Cheers,

Artem






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