Dilute both the Protein and DNA before mixing them at the molar ratio
you require - I would aim to have the protein component at around 1 mg/ml.
Mix, then concentrate together, till you reach the concentration you want.
Antony.
> On Wed, Oct 1, 2008 at 6:52 PM, E rajakumar <[log in to unmask]
> <mailto:[log in to unmask]>> wrote:
>
> Dear All
> Sorry for non-crystallography query. I am working on
> DNA binding protein, while mixing DNA with protein for
> preparing Protein-DNA complex for crystallization,
> protein is precipitating. pI of the protein is 9.3
> and in 15 mM HEPES 7.0, 150mM NaCl and 5% glycerol.
> Concentration of the protein used for mixing with DNA
> is 8 mg/mL. DNA to protein molar ratio is 1.2. Please
> advise me how to prevent precipitation. Is changing of
> buffer pH and adding divalent cation like MgCl2 can
> help in preventing precipitation?
> Thanking You
> Rajakumara
>
>
> E. Rajakumara
> Postdoctoral Fellow
> Strcutural Biology Program
> Memorial Sloan-Kettering Cancer Center
> New York-10021
>
The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP.
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