Hi,
I am trying to crystallize a protein-DNA complex. I purify the protein finally
using gel filtration. I purchase
single stranded complementary oligos (desalting from idtdna.com), mix them up
and make DNA duplex by
heating to 95 degree C and cooling to room temperature. I mix protein and DNA,
concentrate and use it
for crystallization.
I am geting small crystals consistently under a specific condition. These
crystals take up IZIT dye but are
not well shaped. I am not able to improve the size and shape of the crystals
substantially even after
screening with additives (Hampton research).
I suspect that purity of the duplex DNA (presence of unpaired oligos) is
limiting the chances of obtaining
better crystals.
How can I purify the duplex DNA further?
Are there better ways of making protein-DNA complex for crystallization?
If I make the protein –DNA complex and then do the gelfiltration, will the
complex purified so be a better
choice for crystallization?
Thank you
Kumar
Dept. of Biochemistry, Cellular and Molecular Biology,
Walters Life Science, # 406,
University of Tennessee, TN, Knoxville, USA
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