Trouble removing contaminating proteins from amylose resin

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Sat Feb 16 17:40:01 EST 2002


Tried nonionic detergents yet ? Glycerol/ethylene glycol ? Phosphate ? Try
figuring out what the three free-riders are (MS) and add something that'd
get rid of them.

A.G.E.
"John Everett" <kellere at hotmail.com> wrote in message
news:5a7ca595.0202152331.1ebd601c at posting.google.com...
> Newsgroup...
>
>   I have created an amino terminal Maltose Binding Protein (MBP)
> fusion of my pet protein. Unfortunately...  when I bind the fusion
> to an amylose resin, three other proteins invariably bind as well.
> I have shown that the three hitch hikers are binding to my protein
> and not to the resin nor to the MBP.
> I have tried varies amounts of NaCl ( 0 - 1M ), varying pH levels
> (5 - 8). I just can not shake the contaminants off. The contaminants
> stick to the fusion when the fusion is passed through ion exchange
> columns and size exclusions columns.
>
>   I normally wash the resin with 20 column volumes of:
>
>   20  mM Tris-HCL  pH 8.0
>   200 mM NaCl
>   10  mM BME
>
>
>  If any one can offer a wash protocol that may help knock
>  off the contaminating proteins, I would be very appreciative.
>
>   Thanks in advance,
>         John Everett
>
> _______________________________
> UMDNJ | Rutgers University
> kellere at hotmail.com





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