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
