IUBio

6x his tag protein purification

Chen Linan chenlinan at 263.NET
Fri Mar 26 20:57:05 EST 1999


Hi, Robert,

The follow troubleshooting is from my Talon resin manual. Hope it will help.
And would please throw a email here after you succeed in solve the problem? 

Thanks,

Chen Linan

Beijing Medical University
--------------------------
The 6xHis protein may natually form dimers, or may be aggregating into
multimer;both cases may require more stringent elution conditions:
1. For purificaton of dimers using a pH step gradient, prepare an elution
buffer with a lower pH(i.e. pH 5.0-5.3);for purification of dimers using an
imidazole step gradrient,prepare an elution buffer with 100mM
imidazole(final concentration)
2. If the 6xHis protein still resist elution, lower the pH to 4.5 or
increase the imidazole concentration to 200mM(depending on your purification
scheme) and repeat the elution.
3. If protein still resist elution with Elution Buffer B, and repeat the
elution.
4. For really tough elution problems, you can strip the protein off using
100mM EDTA(pH8.0);however, this will remove the cobalt from the resin(and
deposit it in your protein sample).
5. Add 5-10mM beta-ME to reduce disulfide linkage.
6. Supplement buffer with 1% nonionic detergent.
7. Purify 6xHis proteiin under denaturing conditions.



> Dear all
>
> I have just begun using the Talon resin from clontech to purify my his
> tagged protein. On the first run I used 200 mM L-histidine to elute
> from the column. The column turned brown and then this colur eluted
> form the column leaving the column clear not pink as is usual with
> this column resin.  Obviously the metal has leached from the column.
> Is this normal when using histidine to elute from heavy metal affinity
> columns?  Is there a buffer system to use to avoid this?
>
> Thanks
>
> Robert
> R.Seymour at prospect.anprod.csiro.au
>




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