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buffer for His-tag recombinant protein purification - (Jul/12/2006 )

Amersham recommend to use sodium phosphate buffer as binding and washing buffer for His-tag protein purification. So, should I use Na3PO4, or NaH2PO4, or simply could I use PBS instead? Is there any difference between different buffer system?



for his tagged expression you can the QiaExpressionist
I worked with it and found it really good smile.gif


The pH of a phosphate buffer is determined by the balance of Na2HPO4/NaH2PO4 species (Sambrook has a table of pH vs ratios). Straight H3PO4 solution will be too acidic, but if you pH with NaOH you will make a buffer of the right concentration. (You will just make a Na-PO4 solution)

That said, PBS is probably OK, His tags are incredibly tough, I've purified using Tris buffers with no problem. As long as your pH is high enough (7+ is fine) then 6-His will bind to Ni resin.