In vitro translation problems
Edwin ten Dam
etd at st-andrews.ac.uk
Thu Oct 24 09:38:20 EST 1996
In article <326CFC5E.43D9 at mole.bio.cam.ac.uk>, pd1 at mole.bio.cam.ac.uk wrote:
> D.L. Roberts wrote:
> > I have been trying to produce a 35-S labelled protein using Promega's
> > rabbit reticulocyte lysate kit and am having problems as one of the
> > fragments of the active enzyme is approximately 10kD and it is being
> > masked by Haeme proteins which run at about the same position. Has
> > anyone any suggestions on removing the haeme protein or know of a method
> > of preventing it picking up the radiolabel?
> I found that His-bind resin (from Novagen I think, but presumably any
> other Ni-chelate resin?) binds globin remarkably well. Needless to say,
> this wasn't what I was looking for at the time (an exp. involving in
> vitro translation of a his-tagged construct), but I filed it away as
> something that might be useful for other reasons! I guess it requires
> your protein not to bind Ni2+, but if that is the case, perhaps you
> could try gel or centrifugal filtration to separate the 64 kDa globin
> tetramer from your protein (if it's monomeric).
..or maybe try translating it in another system (like wheatgerm)?
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