Hi Sivaraman,
One efficient way to remove nonspecifically associated DNA is to do
polyethyleneimine (PEI) precipitation. Protocols are available online.
Search for PEI precipitation.
Also look up this reference for RNA polymerase purification:
Burgess and Jendrisak. 1975 Biochemistry 14(21):4624-8
It is a great method when many others fail to efficiently remove DNA.
Hope that helps.
Raji
-----------
Raji Edayathumangalam
Joint Research Fellow
Harvard Medical School/
Brigham and Women's Hospital
Brandeis University
On Mar 6, 2010, at 7:23 AM, Sivaraman Padavattan wrote:
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
