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his-tagged protein purification - (Jan/22/2001 )

We have some difficulties to elute a 27 kD His-tagged protein from a nickel purification column We checked the protein is greatly expressed in our extract by western blot but it seems to stich to the beds! Where is the bug? Does a super elute buffer exist?could the extract concentration be too high?Thanks


Why donĀ“t you try a buffer with EDTA (0.5M NaCl, 100mM EDTA, 20mM Tris-HCl pH 7.9). It will quelate Ni2+ and release your protein. You will need to regenerate your column afterwards, but it works.
I hope it helps.