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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