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
Department of Medical Microbiology
Imperial College Medical School at St Mary's
London W2 1PG
Tel: (+44)-(0)171-594 3946
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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