Sivaraman, Unfortunately not all proteins tolerate the high salt concentrations required to dissociated protein:DNA complexes. In these cases we generally use either Benzonase or DNase I in the extraction buffer, or precipitate nucleic acid from the crude extract by adding protamine sulfate.
Interestingly NAD itself has a strong absorbance around 260nm - have you added this to the extraction buffer? >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 ---- Dr Antony W. Oliver Senior Scientist Cancer Research UK DNA Repair Enzymes Group Section of Structural Biology The Institute of Cancer Research 237 Fulham Road LONDON SW3 6JB antony.oli...@icr.ac.uk 020 7153 5571 ---- The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
