Dear All,
I would like to develop a fully automated and "green" purification protocol using an akta. So I plan to elute using a low pH wash, and I need to avoid using EDTA+Ni for column regeneration.
Is there a way to properly wash the IMAC resin without stripping of the metal? For example, an UREA 8M wash, a lower pH wash, 1M Nacl, what else can be used ?
Thank you
You may an analogue of the macromolecule, which has more affinity for the ligand than the original molecule. Usually high ionic strength buffer made up of NaCl or KCl are considered, you may perform elution using calcium chloride or magnesium chloride too if molecule of interest remains bioactive in its presence.
1 M NaCl is not a good idea unless the matrix has remnant charged groups and it will only favor protein binding to the metal ion. Washing of the system between runs is only necessary if you do not get similar results between runs. Eventually a washing is necessary if protein precipitation is ocurring due to non specific binding. This depends on your sample and how it was treated before injecting it to the column. Urea will get rid of precipitated protein but you need to give enough time for urea to work. Also remember that some metal is always stripped during the adsorption process as the system tries to establish equilibrium.
I would like to run multiple samples on the same column. I want to limit cross-contamination but I need to avoid EDTA in between two runs. Any suggestions ?
You can use the Ni-Sepharose excel resin from GE Healthcare. It has a ligand that holds on to the Ni even in 10 mM EDTA according to specifications (and in my experience even in 100 mM). It can be cleaned using 1M NaOH, which should minimize cross contamination. Be aware that it has lower binding capacity than the regular Ni-Sepharose and Chelating Sepharose, so you may need a larger column. If the low pH elution does not work you could look into eluting with ammonium chloride.
I would not use NaOH with metal ions attached as speciation will be perturbed and it could rise to changes in protein adsorption.
I am not an expert on Nickel chemistry, but I have used the Ni-Sepharose excel resin and have not had problems with reduced capacity after CIP with 1M NaOH. It is stated by GE that the resin is chemically stable in 1M NaOH for 24 hours. Knowing how they operate (I worked there for 10 years) they have performed the tests looking at binding capacity, Ni leakage etc.
They also state chemical stability for 1 week in 10 mM HCl, so pH 2 should be fine.
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