a question in protein purification

Daniel Mytelka mytelka at zenith.Berkeley.EDU
Tue May 16 23:32:21 EST 1995

In article <3pb0dv$64n at netnews.upenn.edu>,
yuling luo <yluo at pobox.upenn.edu> wrote:
>Hi, I have produced a recombinant protein that is very basic, but does 
>not bind to cation exchange columns. Its homologous protein is also basic 
>and binds to the cation exchange column well. My current hypothesis is 
>that maybe this recombinant protein binds to DNA or other negatively 
>charged proteins very tight and couldn't be separated. I would appreciate 
>your comments and ideas on how to separate this protein.

If your protein is actually sticking to DNA, you should be able
to coprecipitate it with DNA during a polymin precipitation. Add
polymin to about 0.3%, incubate on ice with occasional pestling
for 30-60 minutes, and spin out the DNA (the supernatant's OD 260
should decrease about 80-90%), and see whether your protein is in the
pellet or not. If it does, a high salt wash may help you strip it
off, and give you partially purified protein (which could then be
further purified on Pcell, DNA-cell, or some similar column).

Dan Mytelka (mytelka at mendel.berkeley.edu)

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