Dear Sivaraman,
in case no one mentioned it, i would simply try ion exchange. DNA will
bind very strongly to anion exchange columns (obviously).
(and probably your protein is basic?)
worked for me many times. and note that if you have a smear on agarose
only tells about the size distr. of your nucleic acid not protein.
with regards to IMAC, to my understanding its not the imidazole but
leaking metal ions that cause the precipitation, this has been
discussed many times... (while of course you are better of changing
the buffer, not a good idea to keep it in high imidazole)
HTH,
Tommi
On Mar 6, 2010, at 7:00 PM, Chun Luo wrote:
> Hi Sivaraman,
>
> NAD+ has A259 peak absorbance. So you may not have that much nucleic
> acids contamination.
>
> However, it is not uncommon to have large amount of nucleic acids
> eluted from Ni columns. Adding our TurboNuclease (http://www.accelagen.com/TurboNuclease-protocol.htm
> ) in lysis buffer can significantly reduce nucleic acids
> contamination and lysate viscosity. TurboNuclease has thousands fold
> higher specific activity than DNaseI. So you only need a little bit.
>
> Many proteins aggregate/precipitate in imidazole. It’s a useful
> precaution to remove imidazole before concentrating proteins. You
> probably concentrated the protein to a minute volume for a 16/60
> column and the concentration could be too high. Try desalting before
> concentrating the protein and try SEC at lower protein concentration.
>
> Good luck!
>
> Chun
>
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf
> Of Sivaraman Padavattan
> Sent: Saturday, March 06, 2010 4:24 AM
> To: [log in to unmask]
> Subject: [ccp4bb] Reg Protein purification
>
> Dear All,
>
> We are trying to purify an enzyme, which requires the co-factor NAD+
> during catalysis by affinity column (Ni-NTA). After induction, the
> bacterial cells were harvested and lysed with 20 mM Tris pH 7.2, 500
> mM NaCl, 5% glycerol, 5 MM B-ME. The resultant supernatant was
> passed through Ni-NTA and bound protein eluted with increasing
> concentration of Imidazole. The eluted proteins was concentrated and
> load onto gelfiltration (Superdex S-75 16/60) column. Our protein
> eluted as a aggregate along with other protein, where A260 was much
> greater than A280, indicative of large fraction of nucleic acid
> contamination. The eluant also appeared as a smear on 1% agarose gel
> electrophoresis. We introduced 1M NaCl in the lysis buffer to
> prevent the nucleic acid interaction. But most of our protein went
> in pellet after cell lysis. We look forward to your valuable
> suggestion to purify the protein free of nucleic acid contamination.
>
> Thanks in advance,
>
> Sivaraman Padavattan
>
>
>
>
Tommi Kajander, Ph.D.
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1
(P.O. Box 65)
00014 Helsinki
Finland
p. +358-9-19158903
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