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?

Koen

_______________________________________________________________

        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
	       http://www.sm.ic.ac.uk/medmicro/home.htm
_______________________________________________________________

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