His tag and second purification step

Koen A.L. De Smet k.desmet at ic.ac.uk
Thu Apr 17 10:34:42 EST 1997

I have a number of proteins with an N-terminal His-tag, expressed at low 
level. Affinity purification on Ni++ enriches them, but it is still only 
10% or so of the total protein. Also, I am convinced that I use enough 
Ni++-NTA (Qiagen), but there still is a lot of protein in the 
flow-through, so it may be that other proteins aspecifically stick to 
the Ni++, preventing my babies to bind.
It has been mentioned several times in this newgroups that a second 
purification step is needed in most cases. But should this be done 
before or after the affinity purification? The feeling I seem to get 
from this newsgroup  seems to be that it is done after the afinity 
purification, but is there a good reason for this?
I would imagine that if I first do an ion-exchange, would I have a 
better and more specific binding of my proteins to the Ni++?

Does anybody have any comments or experience on this? Or should I just 
try it out anyway?



        Dr Koen A.L. De Smet
        Research Fellow
        Department of Medical Microbiology
        Imperial College Medical School at St Mary's
        Norfolk Place
        London W2 1PG
        Great Britain
        Tel: (+44)-(0)171-594 3946  
        Fax: (+44)-(0)171-262-6299   
        Email: k.desmet at sm.ic.ac.uk

"If we knew what it was we were doing,
 it would not be called research, would it?".   --   A.Einstein

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