I am working on protein (lipase enzyme) purification. After using the Sephadex G-25 fine column, which column (Q or DEAE sepharose column) should I use?
What are the buffers should I select?
Thank you
Q and DEAE are anion exchanger resins, meaning that they can bind negatively charged proteins.
Use the sequence of your protein to estimate its pI (the pH at which your protein is neutral, and add two units of pH, (ex : pI = 6, the pH of your buffer must be at 8.0). The elution can be done with a NaCl gradient (from 0-1 M)
Thank you Jorgaq,
I have the crude protein (lipase) along with other proteins. How can I find out the sequence?
Thank you again. waiting for your response
You can try to used any anion exchange resins, any assay the enzyme activity ( lipase) in the wash and elution buffer, to found when the enzyme stay.
Q anion exchangers have quarternary ammonium moities thus we name them as strong anion exchangers. On the other hand, DEAE ( diethylaminoethanol ) has a tertiary amine structure and it is a kind of weaker anion exchanger. DEAEs have narrower pH ranges to be fully ionized rather than Q ones whose nature is always cationic independently from applying pH via mobile phase flow. You can use both for polishing steps if your analyte does not need an extreme pH range for being ionized. Only as minor advice, a certain pH range application for anion exchanger can decrease the matrix interferences at which typically seen in cell culture experiments as acidic nature host cell proteome binding to resins thereby DEAE could be a selection option.
The general application is after intermediate purification with ion exchangers, SEC would be appropriate for polishing and as a final step but that is sure, this sequential app is not mandatory and could be vice versa.
Emir
Venkatesh Mandari you do not know on what lipase are you working? It is from a plasmid or not (with a tag)? If you do know the lipase, go to uniprot and you will find the sequence.
Otherwise, try to enrich your protein as much as possible and send a band of your gel in MS
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