Removing DNA in a cell prep??
wnowatzk at ucs.indiana.edu
Sat Nov 13 09:37:01 EST 1993
I am in the midst of a protein purification project and seem to be losing
protein during the stage at which I remove the DNA. I lyse the cells with
lysozyme (3 mg/g cells) and then sonicate with a probing sonicator to
break up the nucleic acids. The problem is that I can only sonicate small
volumes at a time- so this is a rather tedious process for large preps.
Also I always see ppt of what I assume to be protein and get excessive
foaming. In the past I have tried Polymin P ppt, but titration curves
show that the A(280)/A(260) is pretty much constant, implying that I am
ppt protein as well as N.A. Lastly, I am intentionally avoiding DNase
treatments to keep costs down because I am just working out the prep and
I use lots of cells. I am thinking of trying a Virtis blender or some
other mechanical shearing methods although I am not all that familiar
with these. Any suggestions? Thanks in advance.
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