Hi Sivaraman,
NAD+ has A259 peak absorbance. So you may not have that much nucleic acids contamination. However, it is not uncommon to have large amount of nucleic acids eluted from Ni columns. Adding our TurboNuclease (http://www.accelagen.com/TurboNuclease-protocol.htm) in lysis buffer can significantly reduce nucleic acids contamination and lysate viscosity. TurboNuclease has thousands fold higher specific activity than DNaseI. So you only need a little bit. Many proteins aggregate/precipitate in imidazole. It's a useful precaution to remove imidazole before concentrating proteins. You probably concentrated the protein to a minute volume for a 16/60 column and the concentration could be too high. Try desalting before concentrating the protein and try SEC at lower protein concentration. Good luck! Chun _____ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Sivaraman Padavattan Sent: Saturday, March 06, 2010 4:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reg Protein purification 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
