Dear Kumar,
One often has to try duplexes with many different
ends before getting decent crystals (e.g. 18 for
one project in my lab, even more for others).
Depending on your Kd, you might find that your
complex falls apart during gel filtration.
How long are your oligos? Gel purification
sometimes helps us and sometimes doesn't, but
longer ones (> 20-30nt) are more likely to benefit from it.
Did you check the concentrations yourself before
annealing? (my students get better results when they do).
Can your oligos hairpin? If you have a dimer
that binds a symmetric DNA site, having
individual monomers bound to hairpinned oligos
would certainly make a mess of your xtals.
If you think you have an excess of one single
strand, you could try annealing small amounts at
several different ratios, and check how much is
really duplex on a native agarose or acrylamide gel.
Have you checked your prep for nuclease
contamination? Just incubate some with a
supercoiled plasmid and ~10mM Mg++ for a couple
hours and see if the plasmid stays
supercoiled. This is a beautifully sensitive
assay because cleaving only 1 bond out of
thousands will change the plasmid's mobility -
but bear in mind it will only reveal endonucleases, not exonucleases.
Finally, its always a good idea to pull up a pile
of old papers and skim their methods sections for inspiration.
Good luck!
Phoebe Rice
At 11:01 AM 7/16/2007, you wrote:
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
---------------------------------------------------------------------------------------------------------------------------
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
