The concclusion that you have aggregates in the S75 is not valid in my
opinion. You might simply have a multimer which migrates >~70kDa
totest this hypothesis you should run a S200 or S6 to exclude this
option. What's your molecular weight of the monomer ?
Alternatively you might run a blue native page to confirm your
stochiometry perhaps.
Jürgen
......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/
On Mar 6, 2010, at 7:23, 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
