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 <[email protected]> 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 > > > > > >
