Traditional purification of my protein is performed with Tris-Cl. All of the protocols I’ve found for purifying with the intern-CBD tag uses HEPES. Are they interchangeable for this purpose?
Only if you are not using ion exchange chromatography. Hepes is an acid and has negative charge, it is therefore unsuitable for anion exchange chromatography. Tris, on the other hand, is a base and positively charged, it is unsuitable for cation exchange. In other words, the buffering ion should not bind to the IEC column used, as this could cause sudden changes in pH during elution.
Dr. Buxbaum,
Thank you for your response. After cleavage of the intein-CBD tag I need to concentrate the protein via a phosphocellulose column. I can assume that cleavage of the tag I can either dialyze overnight in a tris buffer or I can simply add enough of the tris buffer to dilute the HEPES buffer. Is that correct? I would rather not dialyze for an extended period of time in that the enzyme is relatively labile.
Scott
If time is an issue, you could use pressure filtration in an Amicon device instead of dialysis. Concentrate the protein, add the new buffer and repeat 2 or 3 times.
Engelbert Buxbaum my cation exchange recommended buffer was 50mM composition but the column I used was old & expired (properly not well stored either). So my protein did not bind and I got it in flow through. Now I want to switch to Mono S column and the recommended buffer is 20mM HEPES, do you think I can dilute it or I still need buffer exchange.
I'd get rid of at least most of the tris, to prevent unwanted pH-changes. A desalting column could be used, or the Amicon cell.
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