GST Protein purification problems
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Tue Nov 27 01:03:22 EST 2001
"Graham Taylor" <graham.taylor at bigfoot.com> wrote in message
news:b1445654.0111190937.13aa0f35 at posting.google.com...
> I'm currently trying to purify a GST tagged protein (100kD) expressed
> in insect cells via baculovirus. Protein is produced and is soluble,
> but... fails to bind to the GST column. The cells were lysed in the
> presence of 5mM EDTA and I performed the binding in the cold room.
> Protein was eluted using glutathione. A small 'smear' of protein of
> increased (!) size was observed in the eluted protein.
> Has anyone else tackled this problem. One final thing, the his tagged
> version of this protein also failed to bind to a Talon column. I'm
> getting tired of all this purification, can anyone help?
I feel your pain. Are you absolutely certain that your tag is in the right
frame? Being out of frame could explain both the increased size(stop codon
isn't being read and termination occurs somewhere downstream) and the lack
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