GST Protein purification problems
graham.taylor at bigfoot.com
Thu Nov 29 14:40:26 EST 2001
"serotonin1" <please_post_replies at newsgroup.com> wrote in message news:<K0GM7.22909$py4.14016772 at news2.nash1.tn.home.com>...
> "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
> of tag.
Thanks for your input!
I've checked the protein with a anti-gst antibody in a western blot
and the tag is present, the same with the 6X His version, both these
proteins contain their respective tags yet fail to bind. I was
thinking of trying sarkosyl to perhaps 'free up' the tag and allow it
to bind to the GST
Has anyone tried this successfully??
All answers gratefully received!
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