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:[email protected]] On Behalf
Of Sivaraman Padavattan
Sent: Saturday, March 06, 2010 4:24 AM
To: [email protected]
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
[email protected]
