secreted eukaryotic HIS-recombinant protein
nospam at nospam.dk
Mon Apr 3 14:42:18 EST 2006
I am producing HIS-tagged secreted proteins in insect cells. There is a
N-terminal v5 and poly HIS for purification. However we are not able to bind
the recombinant protein from the supernatant to nikkel/cobolt columns. Doing
a western blot (on the N-terminal v5) I can see that there is plenty of
protein, but that it runs through the coloumn without binding. We do dialyse
to get rid of HIS from the media, and we also add NaCl to reduce
aggregation. I think that the protein might aggregate in the supernatant so
that it doesn't bind to coloumn.
Does anyone have suggestions on how to prevent/reduce aggregation if that is
the problem. We want the native protein with active disulfide bonds, so
adding for example DTT is not an option! what about Triton for example??
Thanks in advance!
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