Hi,
I have done a hundreds time of denatured Nickel purification. This is my first time encounter this problem.
I was using 6M GuHCl, 300mM NaCl, 50mM Tris pH 8 to wash the column after loading my lysate. Everything looks fine till the moment I added 250mM of Imidazole in my wash buffer to elute my protein. The Nickel started stripping off the column. I couldn't think of any reason for this. I thought the resin was old so I changed to new resin and same thing happen. There is no metal binding domain in my protein.
Please help!
Thanks.
Are you equilibrating the column before with the 250 mM imidazole concentration?
yup. I equilibrated with 1 column volume before adding my lysate. Then wash the column with 500mL, followed by 250mM imidazole elution buffer.
Something must have changed without noticing it. We are dealing with coordination bonds which can only be perturbed by a low pH or the presence of a competing molecule. I would prepare new solutions with new reagents.
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