Mark,

	I agree with Artem in that you may find significant differences in the 
purity of eluants from the 'high fidelity' IMAC resins (I use pre-packed 
HisTrap columns).

	You may also want to have a look at the following reference for a 
comparison of a variety of different affinity tags in terms of purity, 
yield, cost, etc.

Comparison of affinity tags for protein purification. Protein Expr 
Purif. 2005 May;41(1):98-105.

-Andy


On 8/10/2009 11:56 PM, Artem Evdokimov wrote:
> Hi,
> 
> Since you're considering a serious undertaking, it would be good to know
> whether your protein is decently expressed and reasonably characterized in
> its native state - before advising you on the process :)
> 
> If your protein is normally expressed well, is soluble, and not aggregated -
> there's going to be little to no difference between the results of major
> methods used for primary affinity capture. If your protein is not very
> abundant in the lysate, then higher fidelity methods may give you better
> purity - in this case StrepII is somewhat better than GST. You can always
> try the good old Biotin tag - this will however require that your E. coli
> carry excess of BirA (typically supplied from a separate plasmid) as normal
> biotinylation levels in most E. coli strains are fairly low. 
> 
> With 40+ mutants you can expect a certain amount of attrition - some of the
> mutants will not express well and some will not purify well.
> 
> In general there's nothing wrong with single-step IMAC provided that you can
> work out reasonable purification conditions in advance and can assume that
> most of your 40+ mutants behave reasonably well (and close to the test
> case). For outlier mutants there will undoubtedly be issues with
> purification however primary capture method is not likely to fix them since
> it did not cause them in the first place.
> 
> For abundant proteins - if you use a 'high fidelity' IMAC resin such as
> His-SELECT or Talon and if your protein of interest is present (in the
> lysate) in reasonable quantity then you can expect the purity of your
> single-step product to be compatible between GST and IMAC purification.
> StrepII tag is of course a good choice as well.
> 
> Regards,
> 
> Artem
> 
>  "Nothing is built on stone; all is built on sand, but we must build as if
> the sand were stone" 
>  Jorge Luis Borges
>  
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Mark
> Collins
> Sent: Monday, August 10, 2009 10:37 PM
> To: [log in to unmask]
> Subject: [ccp4bb] {Spam?} quick purification?
> 
> Hi All
> 
> A little off topic, but I am thinking about expressing and purifying 40+ 
> mutants, for an assay, at maybe 1mg total protein each.  I'd like the 
> purification to be quick and easy (ie. one step) but cleaner than just a 
> 6his-tag purification.
> 
> Currently, my options are either GST, a longer his tag or a strep-tag.
> 
> Any thoughts on the comparison between GST and strep and Ni purifications 
> would be much appreciated.  OR are there any other (better) high affinity 
> purifications, I've missed, excluding expense Ab resins.
> 
> Thanks,
> Mark Collins
>