Dear Sivaraman
I worked on some protein with DNA contamination recently. Adding DNase
and increasing IMAC wash volume at the same time changed 280/260 ratio
and yield reproducible protein crystals, not high resolution yet....In
case your protein can't stand other treatment...
(Sorry Tommi, I hit the wrong reply button.)
Best,
Zheng (Joe) Zhou
On Sat, Mar 6, 2010 at 8:23 PM, Sivaraman Padavattan
<[log in to unmask]> 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
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