I need to purify His6 tag containing protein. Normally I use lysis buffer containing EDTA and DTT for preparing cell lysate. So can I use same buffer by adding imidazole for chromatographic purification of protein on Ni column?
EDTA is a chelator and will remove the nickel from your column, reducing the amount of protein you can bind to the column (also some of the stripped nickel ions that will float around freely might bind to your protein, which will then be washed away as the nickel is not bound to the column). Most nickel beads/resins/columns can withstand 1-2 mM DTT, but DTT is aggressive and can reduce the nickel, which leads to a brown mess that might ruin your purification. As Saranpal mentioned, TCEP is a better reducing agent in many respects, but it is also horrendously expensive. The most common choice of reducing agent for Ni-NTA tends to be BME (betamercaptoethanol). BME is a less aggressive reducing agent and you can use up to 5 mM and in some cases up to 10 mM BME in your lysis, binding and elution buffers. After the Ni-NTA step, while you dialyse your sample to remove the imidazole you can also remove the BME and change it for DTT if there are any further purification steps. We tend to use TCEP only during the last purification step as it is fairly expensive.
You should not use EDTA in lysis buffer as it will interfere with binding of protein to column. Use phosphate buffer or Tris buffer without EDTA at pH 8. Washing can be done by 100mM EDTA before charging the column with NiSO4. But elution buffer does not require EDTA. Use 200mM of imidazole in Tris buffer to elute your protein. You can make gradient elution with different concentration of imidazole in Tris buffer to check the maximum elution of protein.
Alternatively, you can use TCEP (1-5mM). It is more stable reducing agent than the B-Mercaptoethanol or DTT. I have used upto 5mM on Ni-Column without affecting the binding of my protein to the column. And the best of all, it is odorless.
Good luck
EDTA is a chelator and will remove the nickel from your column, reducing the amount of protein you can bind to the column (also some of the stripped nickel ions that will float around freely might bind to your protein, which will then be washed away as the nickel is not bound to the column). Most nickel beads/resins/columns can withstand 1-2 mM DTT, but DTT is aggressive and can reduce the nickel, which leads to a brown mess that might ruin your purification. As Saranpal mentioned, TCEP is a better reducing agent in many respects, but it is also horrendously expensive. The most common choice of reducing agent for Ni-NTA tends to be BME (betamercaptoethanol). BME is a less aggressive reducing agent and you can use up to 5 mM and in some cases up to 10 mM BME in your lysis, binding and elution buffers. After the Ni-NTA step, while you dialyse your sample to remove the imidazole you can also remove the BME and change it for DTT if there are any further purification steps. We tend to use TCEP only during the last purification step as it is fairly expensive.
Hi all,
I have accidentally added TCEP in bacteria extract and it reaches about 15 mM. Do you think I can load this on a Ni column?
Thank you
Hi all,
although the discussion has been four years ago, just one comment for the researchers looking for that issue: Our INDIGO Ni resin is able to withstand at least 20 mM EDTA and DTT without losing nickel and binding capacity (it even gives better results by adding DTT, don't aks me why...). The product link is
https://cube-biotech.com/products/purification-resins/his-affinity-resins/indigo-ni-agarose-100/368/purecube-100-indigo-ni-agarose
Best, Roland
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