After overnight in-column protease digestion to cleave the GST tag from the protein, the elution always results in a mixture of uncleaved protein, tag, protease and cleaved protein. How do we improve the separation? We have been following the guidelines in the GE manual. Please suggest.
1.If an uncleaved protein is present, I'd assume it did not bind to the resin. Is your column well quilibrated? Did you wash (post loading) and check that you are not seening any unbound intact protein?
2. Do you verify enzyme activity prior to your on-column digestion? If the activity is OK, are you using enough enzyme for the amount of protein?
3. Is the incubation time sufficient?
4. How do you elute the column after incubation with protein and enzyme?
any enzyme reaction depends on concentration of the target substrate and on the enzyme concentration, when you put both concentration on the column you dilute both concentration, another problem is that both proteins are bounded on the column and the enzyme reaction can be done only if both proteins are very close to each other, additional to that both proteins are not moveable on the column and as result the reaction is not repeatable as it should be, and typically different proteins are bounded in different CV-hight of a column therefore most likely both proteins bounded and far away from each other, suggestion - do enzyme reaction in one volume as a classic enzyme reaction (temperature, pH, mixing) then only the column separation.
Hi there,
Normally, o/n digest and elution should be both performed in buffer condition where the fusion protein is in stable interaction with the resin... If it's not the case you have to adjust your protocol. Cleaved protein and protease should be recovered together in elution (the buffer should be free of any GSH in order to avoid GST and uncut fusion elution). To separate cleaved protein from protease I would suggest to pass your sample through an agarose gel activated with a protease inhibitor in order to immobilize the protease used for digest (benzamidine-agarose to trap trypsine or thrombin for instance)
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