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."
https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=17491
Hi,
Adam, you beat me to it!
The instructions that came with the Superose 6 column has very detailed cleaning-in-place (CIP) protocols. You can download the document at the GE website:
https://www.gelifesciences.com/en/us/shop/chromatography/prepacked-columns/size-exclusion/superose-6-increase-small-scale-size-exclusion-chromatography-columns-p-05803?current=29091596#related-documents
If you are not going to use your column for a week or longer, rinse out any salt-containing buffer with water first, and store in 20% absolute ethanol (the ethanol can precipitate salts on the column, so you have to wash with water first).
And as already mentioned, it is possible your protein is degraded. If it has disulfide bonds, it could get nicked, and you don't see the resulting smaller fragment until you run a reducing SDS-PAGE gel. It will still elute at the correct MW from the column.
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