GST pull down vs. 6X his- which method is better?

Vladimir Svetlov svetlov at
Thu Dec 18 16:44:39 EST 1997

In article
<Pine.OSF.3.96.971215103201.20843A-100000 at>, Robert
Livingston <bobl at> wrote:

> Hello,
> I wish to confirm a protein interaction identified in a yeast two-hybrid
> screen using a yeast-independent, in vitro system.  I am considering a GST
> pull down assay or an approach using metal affinity chomatography with a
> 6X his fusion.  The protein interaction I am working with is 
> moderate to weak in binding affinity.  Which of these two methods is more
> robust in detecting weak protein interactions?  Are there better methods I
> should consider?  All responses will be gratefully accepted.

There are people that would swear on their mothers' eyes that one is
infinitely better than the other. Each has its pluses and minuses. One
great thing about His6-tag - Ni-NTA binding is that it is resistant to
denaturing/chaotropic agents. This allows one to use a gradient of, say,
urea to additionally test the stability of interactions/interactors
(Ishihama recently did this to pombe RNA polymerase II to fish out a stable
subassembly of Rpb2/3/11). As is, affinity is not congruent with stability,
and you might need this thing down the road. Other than that, GST is
bigger, which may be a factor for small interactors, and in E. coli it
binds some proteins (chaperones). Robustness-wise both of these methods are
often too robust, picking up something that's not there. As an alternative
(not necessarily better) method I can suggest far-Western. 

Vladimir Svetlov
McArdle Lab for Cancer Research
Dept. Oncology
1400 University Ave.
Madison, WI 53706

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