Hi,
I feel that my Supherose 6 column is contaminated with some protein because I get pretty clean SDS gel bands after Ni NTA but after SEC I see multiple bands on SDS PAGE.
How should I clean the column so that I can be sure that all contamination sticking beforhand on column resin is removed?
Hi there,
I think the best would be to give the pictures of the gels (before and after SEC) for comparison as well as the SEC elution profile to spot what fractions are analyzed by SDS/PAGE. Having multiple bands on SDS/PAGE doesn't automatically mean there are contaminants : for instance partial degradation of a pure protein will result in multiple bands on SDS/PAGE.
As Dominique suggest, you may have partial degradation of your protein after you run it on your column.
What I would like to add is that this could occur because your column may be contaminated with bacteria or other microrganisms. Washing your column with 30 mM sodium azide will kill the live microorganisms. Be careful, however, cause azide is very toxic to humans, as well. I would also consider re-pouring the column, doing the azide wash, and equilibrate your column in buffer in a beaker with several buffer washings/decanting to remove other inorganic material that may be affecting your protein.
"Maintenance and cleaning of size exclusion chromatography columns."
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