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 <[log in to unmask]>
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
>
>
>
>
>
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