Ru Heng,
It is commonly helpful to combine your protein and DNA under
dilute conditions and then concentrate the complex. Combining
concentrated DNA and protein together has a very good chance of
precipitating in my experience. I completely agree that trying
different buffer conditions is a good idea (try to find one that keeps
your protein happy as evaluated by DLS).
Good luck,
-Andy
On 8/13/2009 8:23 AM, Raji Edayathumangalam wrote:
Couple of things, Ru Heng.
1. What buffer conditions is your protein in? Is it similar to the
buffer you describe as using to dissolve your DNA in? In general, you
can even get away with dissolving and annealing the oligos in just Tris etc.
2. Play with buffer conditions, particularly NaCl concentrations.
3. Tweak the protein and DNA ratios. For nucleosomes, we always got
white precipitate if we did not always titrate the DNA to protein ratios
for every individual prep,
I believe optimization of the above parameters would help with the white
precipitate formation.
Hope that helps.
Raji
-----------
Raji Edayathumangalam
Joint Research Fellow
Brigham and Women's Hospital/
Harvard Medical School
Brandeis University
On Aug 13, 2009, at 12:56 AM, ruheng wrote:
Dear CCP4bbers,
I am now working on a DNA binding protein and the purity of the
protein is quite good, however the results of DLS showed that the
protein aggregates terribly in quite a lot of different buffer
conditions I tried and still no crystals can be obtained. So I am
going to co-crystallize the protein in complex with DNA. I synthesized
the oligonucleotides varying different numbers of basepairs to
determine the optimal length which can bound to my protein by EMSA. I
dissoved the oligos in the buffer containing 50mM Tris-HCl, 100mM
NaCl, 10mM MgCl2 and 1mM DTT, pH 7.9 and then annealed the DNA into
the double stranded form at a final concentration of 50uM. When I
performed the EMSA experiment, I mixed the purified protein with the
dsDNA at the molecular ratio approximately 1:1, but white precipitate
was generated as I mixed them.
Does anyone have this kinds of experience when working on DNA binding
proteins and co-crystallizing the protein-DNA complex? Any suggestions
from yours will be appreciated.
Thank you all.
Ru Heng
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