Hi ruheng,
Since you synthesized the oligos, you probably already know if there is
any residual salt or buffer in your oligos. I don't know if that
caused the problem. Sometimes people purify and desalt the oligos
before the annealing step.
Joe
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
搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! 立即试用！
|
