When I try to determine the molecular mass of my highly glycosylated enzyme by size exclusion chromatography on a sepharose matrix I lost the activity. I suppose that the enzyme remains bound to the resin that has a polysaccharide nature. Someone of you know how tyo elute the enzyme? Maybe by a sugar that displaces the enzyme from the resin? Can someone give me a suggestion? Thanks a lot
You didn't say that the protein didn't elute from the column. You said you lost the activity. Did the protein come off the column, but in an inactive state? Try running fractions on SDS-PAGE or doing a protein assay on them to see if the protein came off.
If the enzyme does not elute, try including some salt (150 mM) in the elution buffer to keep the protein from sticking to the column. If your enzyme binds Sepharose, which is made from agarose, then including a high concentration of the D-galactose might compete with binding.
Thanks Adam and Alim,
my enzyme does not elute. I will try with both suggestions. Thanks a lot.
Our enzymes bind specifically to dextran, which is a component of both Sephacryl and Sephadex. To avoid this, we use polyacrylamide gel filtration media, such as the BioGel P-series. Sephadex can be useful for us as an affinity purification step, in which case we elute the enzymes with soluble dextran solutions. Of course, then the enzyme also contains dextran, which can be an issue.
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