Hi,
You can try the following to improve your protein-DNA complex crystals:
1) Try HPLC purified oligos. You can get that from IDTDNA instead of the
standard desalting form. Alternatively, if you have access to a reverse phase
HPLC/column, if you order DNA with Trityl group on, run it on the HPLC, take
trityl group off and run a second time to get pure DNA. But these days with the
advancement of DNA synthesis methods, I think there there's not much of
partial/incomplete product contaminating the samples.
2) Definitely try seeding under different protein concentration, protein:DNA
ratios, etc.
3) Have you tried different protein:DNA ratios? That can have a significant
impact for complex crystals. Also try modifying the DNA ends, overhangs,etc.
You can also try different DNA lengths, design them so that they overlap
end-to-end to complete helical turns,etc.
Refer to:
Tan, S. , et. al JMB 2000,297(4), 947-59.
Thanks,
Debanu.
________________________________
From: CCP4 bulletin board on behalf of bputcha
Sent: Mon 7/16/2007 9:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein-DNA complex for crystallization
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
