In vitro translation problems

Paul Digard pd1 at mole.bio.cam.ac.uk
Tue Oct 22 11:54:57 EST 1996


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?
> 
> Thanks in advance.
> 
> Darren Roberts
> 


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).

Hope this helps

Paul


Dr Paul Digard
Division of Virology                    Tel 01223 336921
Department of Pathology                 Fax 01223 336926
University of Cambridge
Tennis Court Road
Cambridge CB2 1QB
UK



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